Patent application title: Method of Diagnosing, Monitoring, Staging, Imaging and Treating Colon Cancer
Roberto A. Macina (San Jose, CA, US)
Sei-Yu Chen (Foster City, CA, US)
Jason Pluta (Mountain View, CA, US)
Yongming Sun (San Jose, CA, US)
Herve E. Recipon (San Francisco, CA, US)
IPC8 Class: AA61K5110FI
Class name: Drug, bio-affecting and body treating compositions radionuclide or intended radionuclide containing; adjuvant or carrier compositions; intermediate or preparatory compositions attached to antibody or antibody fragment or immunoglobulin; derivative
Publication date: 2008-10-30
Patent application number: 20080267863
Patent application title: Method of Diagnosing, Monitoring, Staging, Imaging and Treating Colon Cancer
Roberto A. Macina
Herve E. Recipon
LICATA & TYRRELL P.C.
Origin: MARLTON, NJ US
IPC8 Class: AA61K5110FI
The present invention provides new methods for detecting, diagnosing,
monitoring, staging, prognosticating, imaging and treating colon cancer.
1. A CSG comprising:(a) a polynucleotide of SEQ ID NO:1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 or a variant
thereof;(b) a protein expressed by a polynucleotide of SEQ ID NO:1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22,
or a variant thereof; or(c) a polynucleotide which is capable of
hybridizing under stringent conditions to the antisense sequence of SEQ
ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21 or 22.
2. A method for diagnosing the presence of colon cancer in a patient comprising:(a) determining levels of the CSG of claim 1 in cells, tissues or bodily fluids in a patient; and(b) comparing the determined levels of CSG with levels of CSG in cells, tissues or bodily fluids from a normal human control, wherein a change in determined levels of CSG in said patient versus normal human control is associated with the presence of colon cancer.
3. A method of diagnosing metastases of colon cancer in a patient comprising:(a) identifying a patient having colon cancer that is not known to have metastasized;(b) determining levels of the CSG of claim 1 in a sample of cells, tissues, or bodily fluid from said patient; and(c) comparing the determined CSG levels with levels of CSG in cells, tissue, or bodily fluid of a normal human control, wherein an increase in determined CSG levels in the patient versus the normal human control is associated with a cancer which has metastasized.
4. A method of staging colon cancer in a patient having colon cancer comprising:(a) identifying a patient having colon cancer;(b) determining levels of the CSG of claim 1 in a sample of cells, tissue, or bodily fluid from said patient; and(c) comparing determined CSG levels with levels of CSG in cells, tissues, or bodily fluid of a normal human control, wherein an increase in determined CSG levels in said patient versus the normal human control is associated with a cancer which is progressing and a decrease in the determined CSG levels is associated with a cancer which is regressing or in remission.
5. A method of monitoring colon cancer in a patient for the onset of metastasis comprising:(a) identifying a patient having colon cancer that is not known to have metastasized;(b) periodically determining levels of the CSG of claim 1 in samples of cells, tissues, or bodily fluid from said patient; and(c) comparing the periodically determined CSG levels with levels of CSG in cells, tissues, or bodily fluid of a normal human control, wherein an increase in any one of the periodically determined CSG levels in the patient versus the normal human control is associated with a cancer which has metastasized.
6. A method of monitoring a change in stage of colon cancer in a patient comprising:(a) identifying a patient having colon cancer;(b) periodically determining levels of the CSG of claim 1 in cells, tissues, or bodily fluid from said patient; and(c) comparing the periodically determined CSG levels with levels of CSG in cells, tissues, or bodily fluid of a normal human control, wherein an increase in any one of the periodically determined CSG levels in the patient versus the normal human control is associated with a cancer which is progressing in stage and a decrease is associated with a cancer which is regressing in stage or in remission.
7. A method of identifying potential therapeutic agents for use in imaging and treating colon cancer comprising screening molecules for an ability to bind to the CSG of claim 1 or decrease expression of the CSG of claim 1 relative to the CSG of claim 1 in the absence of the agent wherein the ability of a molecule to bind to the CSG of claim 1 or decrease expression of the CSG of claim 1 is indicative of the molecule being useful in imaging and treating colon cancer.
8. An antibody which specifically binds a polypeptide encoded by SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22.
9. A method of imaging colon cancer in a patient comprising administering to the patient an antibody of claim 8.
10. The method of claim 9 wherein said antibody is labeled with paramagnetic ions or a radioisotope.
11. A method of treating colon cancer in a patient comprising administering to the patient a molecule which downregulates expression or activity of the CSG of claim 1.
12. A method of inducing an immune response against a target cell expressing the CSG of claim 1 comprising delivering to a human patient an immunogenically stimulatory amount of a CSG protein so that an immune response is mounted against the target cell.
This application is a continuation of U.S. application Ser. No.
11/000,263, filed Nov. 30, 2004, which is a continuation of U.S.
application Ser. No. 09/867,034, filed May 29, 2001, now abandoned, which
claims the benefit of priority from U.S. provisional application Ser. No.
60/207,383 filed May 26, 2000, teachings of each of which are herein
incorporated by reference in their entirety.
FIELD OF THE INVENTION
This invention relates, in part, to newly identified polynucleotides and polypeptides; variants and derivatives of the polynucleotides and polypeptides; processes for making the polynucleotides and the polypeptides, and their variants and derivatives; agonists and antagonists of the polypeptides; and uses of the polynucleotides, polypeptides, variants, derivatives, agonists and antagonists for detecting, diagnosing, monitoring, staging, prognosticating, imaging and treating cancers, particularly colon cancer. In particular, in these and in other regards, the invention relates to colon specific polynucleotides and polypeptides hereinafter referred to as colon specific genes or "CSGs".
BACKGROUND OF THE INVENTION
Cancer of the colon is a highly treatable and often curable disease when localized to the bowel. It is one of the most frequently diagnosed malignancies in the United States as well as the second most common cause of cancer death. Surgery is the primary treatment and results in cure in approximately 50% of patients. However, recurrence following surgery is a major problem and often is the ultimate cause of death.
The prognosis of colon cancer is clearly related to the degree of penetration of the tumor through the bowel wall and the presence or absence of nodal involvement. These two characteristics form the basis for all staging systems developed for this disease. Treatment decisions are usually made in reference to the older Duke's or the Modified Astler-Coller (MAC) classification scheme for staging.
Bowel obstruction and bowel perforation are indicators of poor prognosis in patients with colon cancer. Elevated pretreatment serum levels of carcinoembryonic antigen (CEA) and of carbohydrate antigen 19-9 (CA 19-9) also have a negative prognostic significance.
Age greater than 70 years at presentation is not a contraindication to standard therapies. Acceptable morbidity and mortality, as well as long-term survival, are achieved in this patient population.
Because of the frequency of the disease (approximately 160,000 new cases of colon and rectal cancer per year), the identification of high-risk groups, the demonstrated slow growth of primary lesions, the better survival of early-stage lesions, and the relative simplicity and accuracy of screening tests, screening for colon cancer should be a part of routine care for all adults starting at age 50, especially those with first-degree relatives with colorectal cancer.
Procedures used for detecting, diagnosing, monitoring, staging, and prognosticating colon cancer are of critical importance to the outcome of the patient. For example, patients diagnosed with early colon cancer generally have a much greater five-year survival rate as compared to the survival rate for patients diagnosed with distant metastasized colon cancer. New diagnostic methods which are more sensitive and specific for detecting early colon cancer are clearly needed.
Colon cancer patients are closely monitored following initial therapy and during adjuvant therapy to determine response to therapy and to detect persistent or recurrent disease of metastasis. There is clearly a need for a colon cancer marker which is more sensitive and specific in detecting colon cancer, its recurrence, and progression.
Another important step in managing colon cancer is to determine the stage of the patient's disease. Stage determination has potential prognostic value and provides criteria for designing optimal therapy. Generally, pathological staging of colon cancer is preferable over clinical staging because the former gives a more accurate prognosis. However, clinical staging would be preferred were it at least as accurate as pathological staging because it does not depend on an invasive procedure to obtain tissue for pathological evaluation. Staging of colon cancer would be improved by detecting new markers in cells, tissues, or bodily fluids which could differentiate between different stages of invasion.
Accordingly, there is a great need for more sensitive and accurate methods for the staging of colon cancer in a human to determine whether or not such cancer has metastasized and for monitoring the progress of colon cancer in a human which has not metastasized for the onset of metastasis.
In the present invention, methods are provided for detecting, diagnosing, monitoring, staging, prognosticating, imaging and treating colon cancer via colon specific genes referred to herein as CSGs. For purposes of the present invention, CSG refers, among other things, to native protein expressed by the gene comprising a polynucleotide sequence of SEQ ID NO:1, 2, 3, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22. By "CSG" it is also meant herein polynucleotides which, due to degeneracy in genetic coding, comprise variations in nucleotide sequence as compared to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 but which still encode the same protein. In the alternative, what is meant by CSG as used herein, means the native mRNA encoded by the gene comprising the polynucleotide sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22, levels of the gene comprising the polynucleotide sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22, or levels of a polynucleotide which is capable of hybridizing under stringent conditions to the antisense sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22.
Other objects, features, advantages and aspects of the present invention will become apparent to those of skill in the art from the following description. It should be understood, however, that the following description and the specific examples, while indicating preferred embodiments of the invention are given by way of illustration only. Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following description and from reading the other parts of the present disclosure.
SUMMARY OF THE INVENTION
Toward these ends, and others, it is an object of the present invention to provide CSGs comprising a polynucleotide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22, a protein expressed by a polynucleotide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 or a variant thereof which expresses the protein; or a polynucleotide which is capable of hybridizing under stringent conditions to the antisense sequence of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22.
It is another object of the present invention to provide a method for diagnosing the presence of colon cancer by analyzing for changes in levels of CSG in cells, tissues or bodily fluids compared with levels of CSG in preferably the same cells, tissues, or bodily fluid type of a normal human control, wherein a change in levels of CSG in the patient versus the normal human control is associated with colon cancer.
Further provided is a method of diagnosing metastatic colon cancer in a patient having colon cancer which is not known to have metastasized by identifying a human patient suspected of having colon cancer that has metastasized; analyzing a sample of cells, tissues, or bodily fluid from such patient for CSG; comparing the CSG levels in such cells, tissues, or bodily fluid with levels of CSG in preferably the same cells, tissues, or bodily fluid type of a normal human control, wherein an increase in CSG levels in the patient versus the normal human control is associated with colon cancer which has metastasized.
Also provided by the invention is a method of staging colon cancer in a human which has such cancer by identifying a human patient having such cancer; analyzing a sample of cells, tissues, or bodily fluid from such patient for CSG; comparing CSG levels in such cells, tissues, or bodily fluid with levels of CSG in preferably the same cells, tissues, or bodily fluid type of a normal human control sample, wherein an increase in CSG levels in the patient versus the normal human control is associated with a cancer which is progressing and a decrease in the levels of CSG is associated with a cancer which is regressing or in remission.
Further provided is a method of monitoring colon cancer in a human having such cancer for the onset of metastasis. The method comprises identifying a human patient having such cancer that is not known to have metastasized; periodically analyzing a sample of cells, tissues, or bodily fluid from such patient for CSG; comparing the CSG levels in such cells, tissue, or bodily fluid with levels of CSG in preferably the same cells, tissues, or bodily fluid type of a normal human control sample, wherein an increase in CSG levels in the patient versus the normal human control is associated with a cancer which has metastasized.
Further provided is a method of monitoring the change in stage of colon cancer in a human having such cancer by looking at levels of CSG in a human having such cancer. The method comprises identifying a human patient having such cancer; periodically analyzing a sample of cells, tissues, or bodily fluid from such patient for CSG; comparing the CSG levels in such cells, tissue, or bodily fluid with levels of CSG in preferably the same cells, tissues, or bodily fluid type of a normal human control sample, wherein an increase in CSG levels in the patient versus the normal human control is associated with a cancer which is progressing and a decrease in the levels of CSG is associated with a cancer which is regressing or in remission.
Further provided are methods of designing new therapeutic agents targeted to a CSG for use in imaging and treating colon cancer. For example, in one embodiment, therapeutic agents such as antibodies targeted against CSG or fragments of such antibodies can be used to treat, detect or image localization of CSG in a patient for the purpose of detecting or diagnosing a disease or condition. In this embodiment, an increase in the amount of labeled antibody detected as compared to normal tissue would be indicative of tumor metastases or growth. Such antibodies can be polyclonal, monoclonal, or omniclonal or prepared by molecular biology techniques. The term "antibody", as used herein and throughout the instant specification is also meant to include aptamers and single-stranded oligonucleotides such as those derived from an in vitro evolution protocol referred to as SELEX and well known to those skilled in the art. Antibodies can be labeled with a variety of detectable and therapeutic labels including, but not limited to, radioisotopes and paramagnetic metals. Therapeutic agents such as small molecules and antibodies which decrease the concentration and/or activity of CSG can also be used in the treatment of diseases characterized by overexpression of CSG. Such agents can be readily identified in accordance with teachings herein.
Other objects, features, advantages and aspects of the present invention will become apparent to those of skill in the art from the following description. It should be understood, however, that the following description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only. Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following description and from reading the other parts of the present disclosure.
The following illustrative explanations are provided to facilitate understanding of certain terms used frequently herein, particularly in the examples. The explanations are provided as a convenience and are not limitative of the invention.
ISOLATED means altered "by the hand of man" from its natural state; i.e., that, if it occurs in nature, it has been changed or removed from its original environment, or both.
For example, a naturally occurring polynucleotide or a polypeptide naturally present in a living animal in its natural state is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein. For example, with respect to polynucleotides, the term isolated means that it is separated from the chromosome and cell in which it naturally occurs.
As part of or following isolation, such polynucleotides can be joined to other polynucleotides, such as DNAs, for mutagenesis, to form fusion proteins, and for propagation or expression in a host, for instance. The isolated polynucleotides, alone or joined to other polynucleotides such as vectors, can be introduced into host cells, in culture or in whole organisms. When introduced into host cells in culture or in whole organisms, such DNAs still would be isolated, as the term is used herein, because they would not be in their naturally occurring form or environment. Similarly, the polynucleotides and polypeptides may occur in a composition, such as media formulations, solutions for introduction of polynucleotides or polypeptides, for example, into cells, compositions or solutions for chemical or enzymatic reactions, for instance, which are not naturally occurring compositions, and, therein remain isolated polynucleotides or polypeptides within the meaning of that term as it is employed herein.
OLIGONUCLEOTIDE(S) refers to relatively short polynucleotides. Often the term refers to single-stranded deoxyribonucleotides, but it can refer as well to single- or double-stranded ribonucleotides, RNA:DNA hybrids and double-stranded DNAs, among others.
Oligonucleotides, such as single-stranded DNA probe oligonucleotides, often are synthesized by chemical methods, such as those implemented on automated oligonucleotide synthesizers. However, oligonucleotides can be made by a variety of other methods, including in vitro recombinant DNA-mediated techniques and by expression of DNAs in cells and organisms.
Initially, chemically synthesized DNAs typically are obtained without a 5' phosphate. The 5' ends of such oligonucleotides are not substrates for phosphodiester bond formation by ligation reactions that employ DNA ligases typically used to form recombinant DNA molecules. Where ligation of such oligonucleotides is desired, a phosphate can be added by standard techniques, such as those that employ a kinase and ATP.
The 3' end of a chemically synthesized oligonucleotide generally has a free hydroxyl group and, in the presence of a ligase such as T4 DNA ligase, readily will form a phosphodiester bond with a 5' phosphate of another polynucleotide, such as another oligonucleotide. As is well known, this reaction can be prevented selectively, where desired, by removing the 5' phosphates of the other polynucleotide(s) prior to ligation.
POLYNUCLEOTIDE(S) generally refers to any polyribonucleotide or polydeoxyribonucleotide and is inclusive of unmodified RNA or DNA as well as modified RNA or DNA. Thus, for instance, polynucleotides as used herein refers to, among other things, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, polynucleotide, as used herein, refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide.
As used herein, the term polynucleotide is also inclusive of DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotides" as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein.
It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells, inter alia.
POLYPEPTIDES, as used herein, includes all polypeptides as described below. The basic structure of polypeptides is well known and has been described in innumerable textbooks and other publications in the art. In this context, the term is used herein to refer to any peptide or protein comprising two or more amino acids joined to each other in a linear chain by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. It will be appreciated that polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids, and that many amino acids, including the terminal amino acids, may be modified in a given polypeptide, either by natural processes such as processing and other post-translational modifications, or by chemical modification techniques which are well known to the art. Even the common modifications that occur naturally in polypeptides are too numerous to list exhaustively here, but they are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature, and they are well known to those of skill in the art.
Modifications which may be present in polypeptides of the present invention include, to name an illustrative few, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
Such modifications are well known to those of skill and have been described in great detail in the scientific literature. Several particularly common modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation are described in most basic texts, such as, for instance PROTEINS STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993). Many detailed reviews are available on this subject, such as, for example, those provided by Wold, F., Posttranslational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York (1983); Seifter et al., Analysis for protein modifications and nonprotein cofactors, Meth. Enzymol. 182: 626-646 (1990) and Rattan et al., Protein Synthesis: Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663: 48-62 (1992).
It will be appreciated that the polypeptides of the present invention are not always entirely linear. Instead, polypeptides may be branched as a result of ubiquitination, and they may be circular, with or without branching, generally as a result of posttranslation events including natural processing event and events brought about by human manipulation which do not occur naturally. Circular, branched and branched circular polypeptides may be synthesized by non-translation natural processes and by entirely synthetic methods, as well.
Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. In fact, blockage of the amino and/or carboxyl group in a polypeptide by a covalent modification is common in naturally occurring and synthetic polypeptides and such modifications may be present in polypeptides of the present invention, as well. For instance, the amino terminal residue of polypeptides made in E. coli, prior to proteolytic processing, almost invariably will be N-formylmethionine.
The modifications that occur in a polypeptide often will be a function of how it is made. For polypeptides made by expressing a cloned gene in a host, for instance, the nature and extent of the modifications, in large part, will be determined by the host cell posttranslational modification capacity and the modification signals present in the polypeptide amino acid sequence. For instance, as is well known, glycosylation often does not occur in bacterial hosts such as E. coli. Accordingly, when glycosylation is desired, a polypeptide can be expressed in a glycosylating host, generally a eukaryotic cell. Insect cells often carry out the same posttranslational glycosylations as mammalian cells. Thus, insect cell expression systems have been developed to express efficiently mammalian proteins having native patterns of glycosylation, inter alia. Similar considerations apply to other modifications.
It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications.
In general, as used herein, the term polypeptide encompasses all such modifications, particularly those that are present in polypeptides synthesized by expressing a polynucleotide in a host cell.
VARIANT(S) of polynucleotides or polypeptides, as the term is used herein, are polynucleotides or polypeptides that differ from a reference polynucleotide or polypeptide, respectively.
With respect to variant polynucleotides, differences are generally limited so that the nucleotide sequences of the reference and the variant are closely similar overall and, in many regions, identical. Thus, changes in the nucleotide sequence of the variant may be silent. That is, they may not alter the amino acids encoded by the polynucleotide. Where alterations are limited to silent changes of this type a variant will encode a polypeptide with the same amino acid sequence as the reference. Alternatively, changes in the nucleotide sequence of the variant may alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Such nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence.
With respect to variant polypeptides, differences are generally limited so that the sequences of the reference and the variant are closely similar overall and, in many region, identical. For example, a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions, fusions and truncations, which may be present in any combination.
RECEPTOR MOLECULE, as used herein, refers to molecules which bind or interact specifically with CSG polypeptides of the present invention and is inclusive not only of classic receptors, which are preferred, but also other molecules that specifically bind to or interact with polypeptides of the invention (which also may be referred to as "binding molecules" and "interaction molecules," respectively and as "CSG binding or interaction molecules". Binding between polypeptides of the invention and such molecules, including receptor or binding or interaction molecules may be exclusive to polypeptides of the invention, which is very highly preferred, or it may be highly specific for polypeptides of the invention, which is highly preferred, or it may be highly specific to a group of proteins that includes polypeptides of the invention, which is preferred, or it may be specific to several groups of proteins at least one of which includes polypeptides of the invention.
Receptors also may be non-naturally occurring, such as antibodies and antibody-derived reagents that bind to polypeptides of the invention.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to novel colon specific polypeptides and polynucleotides, referred to herein as CSGs, among other things, as described in greater detail below.
In accordance with one aspect of the present invention, there are provided isolated CSG polynucleotides which encode CSG polypeptides.
Using the information provided herein, such as the polynucleotide sequences set out in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 a polynucleotide of the present invention encoding a CSG may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using mRNA from cells of a human tumor as starting material.
Polynucleotides of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced by chemical synthetic techniques or by a combination thereof. The DNA may be double-stranded or single-stranded. Single-stranded DNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand.
The coding sequence which encodes the polypeptides may be identical to the coding sequence of the polynucleotides of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22. It also may be a polynucleotide with a different sequence, which, as a result of the redundancy (degeneracy) of the genetic code, encodes the same polypeptides as encoded by SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22.
Polynucleotides of the present invention such as SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 which encode these polypeptides may include, but are not limited to the coding sequence for the mature polypeptide, by itself; the coding sequence for the mature polypeptide and additional coding sequences, such as those encoding a leader or secretory sequence, such as a pre-, or pro- or prepro-protein sequence; the coding sequence of the mature polypeptide, with or without the aforementioned additional coding sequences, together with additional, non-coding sequences, including for example, but not limited to introns and non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing including, for example, splicing and polyadenylation signals, ribosome binding and stability of mRNA, additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities. Thus, for instance, the polypeptide may be fused to a marker sequence, such as a peptide, which facilitates purification of the fused polypeptide. In certain preferred embodiments of this aspect of the invention, the marker sequence is a hexa-histidine peptide, such as the tag provided in the pQE vector (Qiagen, Inc., among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci., USA 86: 821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. The HA tag corresponds to an epitope derived of influenza hemagglutinin protein, which has been described by Wilson et al., Cell 37: 767 (1984), for instance.
In accordance with the foregoing, the term "polynucleotide encoding a polypeptide" as used herein encompasses polynucleotides which include a sequence encoding a polypeptide of the present invention. The term encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (for example, interrupted by introns) together with additional regions, that also may contain coding and/or non-coding sequences.
The present invention further relates to variants of the herein above described polynucleotides which encode for fragments, analogs and derivatives of the polypeptides. A variant of the polynucleotide may be a naturally occurring variant such as a naturally occurring allelic variant, or it may be a variant that is not known to occur naturally. Such non-naturally occurring variants of the polynucleotide may be made by mutagenesis techniques, including those applied to polynucleotides, cells or organisms.
Among variants in this regard are variants that differ from the aforementioned polynucleotides by nucleotide substitutions, deletions or additions. The substitutions, deletions or additions may involve one or more nucleotides. The variants may be altered in coding or non-coding regions or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions.
Further particularly preferred in this regard are CSG polynucleotides encoding variants, analogs, derivatives and fragments, and variants, analogs and derivatives of the fragments, in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, deleted or added, in any combination. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the CSGS. Also especially preferred in this regard are conservative substitutions.
Further preferred embodiments of the invention are CSG polynucleotides that are at least 70% identical to a polynucleotide comprising SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22, and polynucleotides which are complementary to such polynucleotides. Alternatively, most highly preferred are CSG polynucleotides that comprise a region that is at least 80% identical to a polynucleotide comprising SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22. In this regard, CSG polynucleotides at least 90% identical to the same are particularly preferred, and among these particularly preferred CSG polynucleotides, those with at least 95% are especially preferred. Furthermore, those with at least 97% are highly preferred among those with at least 95%, and among these those with at least 98% and at least 99% are particularly highly preferred, with at least 99% being the more preferred.
Particularly preferred embodiments in this respect, moreover, are polynucleotides which encode polypeptides which retain substantially the same biological function or activity as the mature polypeptides encoded by the human cDNA.
The present invention further relates to polynucleotides that hybridize to the herein above-described CSG sequences. In this regard, the present invention especially relates to polynucleotides which hybridize under stringent conditions to the herein above-described polynucleotides. As herein used, the term "stringent conditions" means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences.
As discussed additionally herein regarding polynucleotide assays of the invention, for instance, polynucleotides of the invention as discussed above, may be used as a hybridization probe for cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding CSGs and to isolate cDNA and genomic clones of other genes that have a high sequence similarity to the CSGs. Such probes generally will comprise at least 15 bases. Preferably, such probes will have at least 30 bases and may have at least 50 bases.
For example, the coding region of the CSGs may be isolated by screening using the known DNA sequence to synthesize an oligonucleotide probe. A labeled oligonucleotide having a sequence complementary to that of a gene of the present invention is then used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.
The polynucleotides and polypeptides of the present invention may be employed as research reagents and materials for discovery of treatments and diagnostics to human disease, as further discussed herein relating to polynucleotide assays, inter alia.
The polynucleotides may encode a polypeptide which is the mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mature polypeptide (when the mature form has more than one polypeptide chain, for instance). Such sequences may play a role in processing of a protein from precursor to a mature form, may facilitate/protein trafficking, may prolong or shorten protein half-life or may facilitate manipulation of a protein for assay or production, among other things. As generally is the case in situ, the additional amino acids may be processed away from the mature protein by cellular enzymes.
A precursor protein, having the mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide. When prosequences are removed such inactive precursors generally are activated. Some or all of the prosequences may be removed before activation. Generally, such precursors are called proproteins.
In sum, a polynucleotide of the present invention may encode a mature protein, a mature protein plus a leader sequence (which may be referred to as a preprotein), a precursor of a mature protein having one or more prosequences which are not the leader sequences of a preprotein, or a preproprotein, which is a precursor to a proprotein, having a leader sequence and one or more prosequences, which generally are removed during processing steps that produce active and mature forms of the polypeptide.
The present invention further relates to CSG polypeptides. The invention also relates to fragments, analogs and derivatives of these polypeptides. The terms "fragment," "derivative" and "analog" when referring to the polypeptides, means a polypeptide which retains essentially the same biological function or activity as such polypeptides. Thus, an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.
The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide. In certain preferred embodiments it is a recombinant polypeptide.
The fragment, derivative or analog of the polypeptide may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.
Among preferred variants are those that vary from a reference by conservative amino acid substitutions. Such substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu and Ile; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gln, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe, Tyr.
The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
The polypeptides of the present invention include the native protein expressed by the gene comprising a polynucleotide sequence of SEQ ID NO:1, 2, 3, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 as well as polypeptides which have at least 75% similarity (preferably at least 75% identity) to these polypeptides and more preferably at least 90% similarity (more preferably at least 90% identity) to these polypeptides and still more preferably at least 95% similarity (still more preferably at least 95% identity) to these polypeptides and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids.
As known in the art "similarity" between two polypeptides is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide sequence with that of a second polypeptide.
Fragments or portions of the polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, the fragments may be employed as intermediates for producing the full-length polypeptides. Fragments or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotides of the present invention.
Also among preferred embodiments of this aspect of the present invention are polypeptides comprising fragments of native protein expressed by the gene comprising a polynucleotide sequence of SEQ ID NO:1, 2, 3, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22.
In this regard a fragment is a polypeptide having an amino acid sequence that entirely is the same as part but not all of the amino acid sequence of the aforementioned polypeptides and variants or derivatives thereof.
Such fragments may be "free-standing," i.e., not part of or fused to other amino acids or polypeptides, or they may be comprised within a larger polypeptide of which they form a part or region. When comprised within a larger polypeptide, the presently discussed fragments most preferably form a single continuous region. However, several fragments may be comprised within a single larger polypeptide. For instance, certain preferred embodiments relate to a fragment of a polypeptide of the present invention comprised within a precursor polypeptide designed for expression in a host and having heterologous pre and pro-polypeptide regions fused to the amino terminus of the polypeptide fragment and an additional region fused to the carboxyl terminus of the fragment. Therefore, fragments in one aspect of the meaning intended herein, refers to the portion or portions of a fusion polypeptide or fusion protein derived from a polypeptide of the present invention.
As representative examples of polypeptide fragments of the invention, there may be mentioned those which have from about 15 to about 139 amino acids.
In this context "about" includes the particularly recited range and ranges larger or smaller by several, a few, 5, 4, 3, 2 or 1 amino acid at either extreme or at both extremes. Highly preferred in this regard are the recited ranges plus or minus as many as 5 amino acids at either or at both extremes. Particularly highly preferred are the recited ranges plus or minus as many as 3 amino acids at either or at both the recited extremes. Especially preferred are ranges plus or minus 1 amino acid at either or at both extremes or the recited ranges with no additions or deletions. Most highly preferred of all in this regard are fragments from about 15 to about 45 amino acids.
Among especially preferred fragments of the invention are truncation mutants of native protein expressed by the gene comprising a polynucleotide sequence of SEQ ID NO:1, 2, 3, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22. Truncation mutants include polypeptides having the amino acid sequence of native protein expressed by the gene comprising a polynucleotide sequence of SEQ ID NO:1, 2, 3, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22, or variants or derivatives thereof, except for deletion of a continuous series of residues (that is, a continuous region, part or portion) that includes the amino terminus, or a continuous series of residues that includes the carboxyl terminus or, as in double truncation mutants, deletion of two continuous series of residues, one including the amino terminus and one including the carboxyl terminus. Fragments having the size ranges set out about also are preferred embodiments of truncation fragments, which are especially preferred among fragments generally.
Also preferred in this aspect of the invention are fragments characterized by structural or functional attributes of native protein expressed by the gene comprising a polynucleotide sequence of SEQ ID NO:1, 2, 3, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22. Preferred embodiments of the invention in this regard include fragments that comprise alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-sheet and beta-sheet-forming regions ("beta-regions"), turn and turn-forming regions ("turn-regions"), coil and coil-forming regions ("coil-regions"), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions of native protein expressed by the gene comprising a polynucleotide sequence of SEQ ID NO:1, 2, 3, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22.
Among highly preferred fragments in this regard are those that comprise regions of native protein expressed by the gene comprising a polynucleotide sequence of SEQ ID NO:1, 2, 3, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 that combine several structural features, such as several of the features set out above. In this regard, the regions defined by the residues of native protein expressed by the gene comprising a polynucleotide sequence of SEQ ID NO:1, 2, 3, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22, which all are characterized by amino acid compositions highly characteristic of turn-regions, hydrophilic regions, flexible-regions, surface-forming regions, and high antigenic index-regions, are especially highly preferred regions. Such regions may be comprised within a larger polypeptide or may be by themselves a preferred fragment of the present invention, as discussed above. It will be appreciated that the term "about" as used in this paragraph has the meaning set out above regarding fragments in general.
Further preferred regions are those that mediate activities of native protein expressed by the gene comprising a polynucleotide sequence of SEQ ID NO:1, 2, 3, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22. Most highly preferred in this regard are fragments that have a chemical, biological or other activity of native protein expressed by the gene comprising a polynucleotide sequence of SEQ ID NO:1, 2, 3, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Highly preferred in this regard are fragments that contain regions that are homologs in sequence, or in position, or in both sequence and to active regions of related polypeptides, such as the native protein expressed by the gene comprising a polynucleotide sequence of SEQ ID NO:1, 2, 3, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22, and which include colon specific-binding proteins. Among particularly preferred fragments in these regards are truncation mutants, as discussed above.
It will be appreciated that the invention also relates to, among others, polynucleotides encoding the aforementioned fragments, polynucleotides that hybridize to polynucleotides encoding the fragments, particularly those that hybridize under stringent conditions, and polynucleotides, such as PCR primers, for amplifying polynucleotides that encode the fragments. In these regards, preferred polynucleotides are those that correspondent to the preferred fragments, as discussed above.
The present invention also relates to diagnostic assays and methods, both quantitative and qualitative for detecting, diagnosing, monitoring, staging and prognosticating cancers by comparing levels of CSG in a human patient with those of LSG in a normal human control. For purposes of the present invention, what is meant by CSG levels is, among other things, native protein expressed by the gene comprising a polynucleotide sequence of SEQ ID NO:1, 2, 3, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22. By "CSG" it is also meant herein polynucleotides which, due to degeneracy in genetic coding, comprise variations in nucleotide sequence as compared to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 but which still encode the same protein. In the alternative, what is meant by CSG as used herein, means the native mRNA encoded by the gene comprising the polynucleotide sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22, levels of the gene comprising the polynucleotide sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22, or levels of a polynucleotide which is capable of hybridizing under stringent conditions to the antisense sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22. Such levels are preferably determined in at least one of cells, tissues and/or bodily fluids, including determination of normal and abnormal levels. Thus, for instance, a diagnostic assay in accordance with the invention for diagnosing overexpression of CSG protein compared to normal control bodily fluids, cells, or tissue samples may be used to diagnose the presence of colon cancer.
All the methods of the present invention may optionally include determining the levels of other cancer markers as well as CSG. Other cancer markers, in addition to CSG, useful in the present invention will depend on the cancer being tested and are known to those of skill in the art.
The present invention provides methods for diagnosing the presence of colon cancer by analyzing for changes in levels of CSG in cells, tissues or bodily fluids compared with levels of CSG in cells, tissues or bodily fluids of preferably the same type from a normal human control, wherein an increase in levels of CSG in the patient versus the normal human control is associated with the presence of colon cancer.
Without limiting the instant invention, typically, for a quantitative diagnostic assay a positive result indicating the patient being tested has cancer is one in which cells, tissues or bodily fluid levels of the cancer marker, such as CSG, are at least two times higher, and most preferably are at least five times higher, than in preferably the same cells, tissues or bodily fluid of a normal human control.
The present invention also provides a method of diagnosing metastatic colon cancer in a patient having colon cancer which has not yet metastasized for the onset of metastasis. In the method of the present invention, a human cancer patient suspected of having colon cancer which may have metastasized (but which was not previously known to have metastasized) is identified. This is accomplished by a variety of means known to those of skill in the art.
In the present invention, determining the presence of CSG levels in cells, tissues or bodily fluid, is particularly useful for discriminating between colon cancer which has not metastasized and colon cancer which has metastasized. Existing techniques have difficulty discriminating between colon cancer which has metastasized and colon cancer which has not metastasized and proper treatment selection is often dependent upon such knowledge.
In the present invention, the cancer marker levels measured in such cells, tissues or bodily fluid is CSG, and are compared with levels of CSG in preferably the same cells, tissue or bodily fluid type of a normal human control. That is, if the cancer marker being observed is just CSG in serum, this level is preferably compared with the level of CSG in serum of a normal human control. An increase in the CSG in the patient versus the normal human control is associated with colon cancer which has metastasized.
Without limiting the instant invention, typically, for a quantitative diagnostic assay a positive result indicating the cancer in the patient being tested or monitored has metastasized is one in which cells, tissues or bodily fluid levels of the cancer marker, such as CSG, are at least two times higher, and most preferably are at least five times higher, than in preferably the same cells, tissues or bodily fluid of a normal patient.
Normal human control as used herein includes a human patient without cancer and/or non cancerous samples from the patient; in the methods for diagnosing or monitoring for metastasis, normal human control may preferably also include samples from a human patient that is determined by reliable methods to have colon cancer which has not metastasized.
The invention also provides a method of staging colon cancer in a human patient. The method comprises identifying a human patient having such cancer and analyzing cells, tissues or bodily fluid from such human patient for CSG. The CSG levels determined in the patient are then compared with levels of CSG in preferably the same cells, tissues or bodily fluid type of a normal human control, wherein an increase in CSG levels in the human patient versus the normal human control is associated with a cancer which is progressing and a decrease in the levels of CSG (but still increased over true normal levels) is associated with a cancer which is regressing or in remission.
Further provided is a method of monitoring colon cancer in a human patient having such cancer for the onset of metastasis. The method comprises identifying a human patient having such cancer that is not known to have metastasized; periodically analyzing cells, tissues or bodily fluid from such human patient for LSG; and comparing the CSG levels determined in the human patient with levels of CSG in preferably the same cells, tissues or bodily fluid type of a normal human control, wherein an increase in CSG levels in the human patient versus the normal human control is associated with a cancer which has metastasized. In this method, normal human control samples may also include prior patient samples.
Further provided by this invention is a method of monitoring the change in stage of colon cancer in a human patient having such cancer. The method comprises identifying a human patient having such cancer; periodically analyzing cells, tissues or bodily fluid from such human patient for LSG; and comparing the CSG levels determined in the human patient with levels of CSG in preferably the same cells, tissues or bodily fluid type of a normal human control, wherein an increase in CSG levels in the human patient versus the normal human control is associated with a cancer which is progressing in stage and a decrease in the levels of CSG is associated with a cancer which is regressing in stage or in remission. In this method, normal human control samples may also include prior patient samples.
Monitoring a patient for onset of metastasis is periodic and preferably done on a quarterly basis. However, this may be done more or less frequently depending on the cancer, the particular patient, and the stage of the cancer.
Prognostic Testing and Clinical Trial Monitoring
The methods described herein can further be utilized as prognostic assays to identify subjects having or at risk of developing a disease or disorder associated with increased levels of CSG. The present invention provides a method in which a test sample is obtained from a human patient and CSG is detected. The presence of higher CSG levels as compared to normal human controls is diagnostic for the human patient being at risk for developing cancer, particularly colon cancer.
The effectiveness of therapeutic agents to decrease expression or activity of the CSGs of the invention can also be monitored by analyzing levels of expression of the CSGs in a human patient in clinical trials or in in vitro screening assays such as in human cells. In this way, the gene expression pattern can serve as a marker, indicative of the physiological response of the human patient, or cells as the case may be, to the agent being tested.
Detection of Genetic Lesions or Mutations
The methods of the present invention can also be used to detect genetic lesions or mutations in CSG, thereby determining if a human with the genetic lesion is at risk for colon cancer or has colon cancer. Genetic lesions can be detected, for example, by ascertaining the existence of a deletion and/or addition and/or substitution of one or more nucleotides from the CSGs of this invention, a chromosomal rearrangement of CSG, aberrant modification of CSG (such as of the methylation pattern of the genomic DNA), the presence of a non-wild type splicing pattern of a mRNA transcript of CSG, allelic loss of CSG, and/or inappropriate post-translational modification of CSG protein. Methods to detect such lesions in the CSG of this invention are known to those of skill in the art.
Assay techniques that can be used to determine levels of gene expression (including protein levels), such as CSG of the present invention, in a sample derived from a patient are well known to those of skill in the art. Such assay methods include, without limitation, radioimmunoassays, reverse transcriptase PCR (RT-PCR) assays, immunohistochemistry assays, in situ hybridization assays, competitive-binding assays, Western Blot analyses, ELISA assays and proteomic approaches: two-dimensional gel electrophoresis (2D electrophoresis) and non-gel based approaches such as mass spectrometry or protein interaction profiling. Among these, ELISAs are frequently preferred to diagnose a gene's expressed protein in biological fluids.
An ELISA assay initially comprises preparing an antibody, if not readily available from a commercial source, specific to CSG, preferably a monoclonal antibody. In addition a reporter antibody generally is prepared which binds specifically to CSG. The reporter antibody is attached to a detectable reagent such as radioactive, fluorescent or enzymatic reagent, for example horseradish peroxidase enzyme or alkaline phosphatase.
To carry out the ELISA, antibody specific to CSG is incubated on a solid support, e.g. a polystyrene dish, that binds the antibody. Any free protein binding sites on the dish are then covered by incubating with a non-specific protein such as bovine serum albumin. Next, the sample to be analyzed is incubated in the dish, during which time CSG binds to the specific antibody attached to the polystyrene dish. Unbound sample is washed out with buffer. A reporter antibody specifically directed to CSG and linked to a detectable reagent such as horseradish peroxidase is placed in the dish resulting in binding of the reporter antibody to any monoclonal antibody bound to CSG. Unattached reporter antibody is then washed out. Reagents for peroxidase activity, including a calorimetric substrate are then added to the dish. Immobilized peroxidase, linked to CSG antibodies, produces a colored reaction product. The amount of color developed in a given time period is proportional to the amount of CSG protein present in the sample. Quantitative results typically are obtained by reference to a standard curve.
A competition assay can also be employed wherein antibodies specific to CSG are attached to a solid support and labeled CSG and a sample derived from the host are passed over the solid support. The amount of label detected which is attached to the solid support can be correlated to a quantity of CSG in the sample.
Using all or a portion of a nucleic acid sequence of CSG of the present invention as a hybridization probe, nucleic acid methods can also be used to detect CSG mRNA as a marker for colon cancer. Polymerase chain reaction (PCR) and other nucleic acid methods, such as ligase chain reaction (LCR) and nucleic acid sequence based amplification (NASBA), can be used to detect malignant cells for diagnosis and monitoring of various malignancies. For example, reverse-transcriptase PCR (RT-PCR) is a powerful technique which can be used to detect the presence of a specific mRNA population in a complex mixture of thousands of other mRNA species. In RT-PCR, an mRNA species is first reverse transcribed to complementary DNA (cDNA) with use of the enzyme reverse transcriptase; the cDNA is then amplified as in a standard PCR reaction. RT-PCR can thus reveal by amplification the presence of a single species of mRNA. Accordingly, if the mRNA is highly specific for the cell that produces it, RT-PCR can be used to identify the presence of a specific type of cell.
Hybridization to clones or oligonucleotides arrayed on a solid support (i.e. gridding) can be used to both detect the expression of and quantitate the level of expression of that gene. In this approach, a cDNA encoding the LSG gene is fixed to a substrate. The substrate may be of any suitable type including but not limited to glass, nitrocellulose, nylon or plastic. At least a portion of the DNA encoding the CSG gene is attached to the substrate and then incubated with the analyte, which may be RNA or a complementary DNA (cDNA) copy of the RNA, isolated from the tissue of interest. Hybridization between the substrate bound DNA and the analyte can be detected and quantitated by several means including but not limited to radioactive labeling or fluorescence labeling of the analyte or a secondary molecule designed to detect the hybrid. Quantitation of the level of gene expression can be done by comparison of the intensity of the signal from the analyte compared with that determined from known standards. The standards can be obtained by in vitro transcription of the target gene, quantitating the yield, and then using that material to generate a standard curve.
Of the proteomic approaches, 2D electrophoresis is a technique well known to those in the art. Isolation of individual proteins from a sample such as serum is accomplished using sequential separation of proteins by different characteristics usually on polyacrylamide gels. First, proteins are separated by size using an electric current. The current acts uniformly on all proteins, so smaller proteins move farther on the gel than larger proteins. The second dimension applies a current perpendicular to the first and separates proteins not on the basis of size but on the specific electric charge carried by each protein. Since no two proteins with different sequences are identical on the basis of both size and charge, the result of a 2D separation is a square gel in which each protein occupies a unique spot. Analysis of the spots with chemical or antibody probes, or subsequent protein microsequencing can reveal the relative abundance of a given protein and the identity of the proteins in the sample.
The above tests can be carried out on samples derived from a variety of cells, bodily fluids and/or tissue extracts such as homogenates or solubilized tissue obtained from a patient. Tissue extracts are obtained routinely from tissue biopsy and autopsy material. Bodily fluids useful in the present invention include blood, urine, saliva or any other bodily secretion or derivative thereof. By blood it is meant to include whole blood, plasma, serum or any derivative of blood.
In Vivo Targeting of CSG/Colon Cancer Therapy
Identification of this CSG is also useful in the rational design of new therapeutics for imaging and treating cancers, and in particular colon cancer. For example, in one embodiment, antibodies which specifically bind to CSG can be raised and used in vivo in patients suspected of suffering from colon cancer. Antibodies which specifically bind CSG can be injected into a patient suspected of having colon cancer for diagnostic and/or therapeutic purposes. Thus, another aspect of the present invention provides for a method for preventing the onset and treatment of colon cancer in a human patient in need of such treatment by administering to the patient an effective amount of antibody. By "effective amount" it is meant the amount or concentration of antibody needed to bind to the target antigens expressed on the tumor to cause tumor shrinkage for surgical removal, or disappearance of the tumor. The binding of the antibody to the overexpressed CSG is believed to cause the death of the cancer cell expressing such CSG. The preparation and use of antibodies for in vivo diagnosis and treatment is well known in the art. For example, antibody-chelators labeled with Indium-111 have been described for use in the radioimmunoscintographic imaging of carcinoembryonic antigen expressing tumors (Sumerdon et al. Nucl. Med. Biol. 1990 17:247-254). In particular, these antibody-chelators have been used in detecting tumors in patients suspected of having recurrent colorectal cancer (Griffin et al. J. Clin. Ono. 1991 9:631-640). Antibodies with paramagnetic ions as labels for use in magnetic resonance imaging have also been described (Lauffer, R.B. Magnetic Resonance in Medicine 1991 22:339-342). Antibodies directed against CSG can be used in a similar manner. Labeled antibodies which specifically bind CSG can be injected into patients suspected of having colon cancer for the purpose of diagnosing or staging of the disease status of the patient. The label used will be selected in accordance with the imaging modality to be used. For example, radioactive labels such as Indium-111, Technetium-99m or Iodine-131 can be used for planar scans or single photon emission computed tomography (SPECT). Positron emitting labels such as Fluorine-19 can be used in positron emission tomography. Paramagnetic ions such as Gadlinium (III) or Manganese (II) can be used in magnetic resonance imaging (MRI). Presence of the label, as compared to imaging of normal tissue, permits determination of the spread of the cancer. The amount of label within an organ or tissue also allows determination of the presence or absence of cancer in that organ or tissue.
Antibodies which can be used in in vivo methods include polyclonal, monoclonal and omniclonal antibodies and antibodies prepared via molecular biology techniques. Antibody fragments and aptamers and single-stranded oligonucleotides such as those derived from an in vitro evolution protocol referred to as SELEX and well known to those skilled in the art can also be used.
The present invention also provides methods for identifying modulators which bind to CSG protein or have a modulatory effect on the expression or activity of CSG protein. Modulators which decrease the expression or activity of CSG protein are believed to be useful in treating colon cancer. Such screening assays are known to those of skill in the art and include, without limitation, cell-based assays and cell free assays.
Small molecules predicted via computer imaging to specifically bind to regions of CSG can also be designed, synthesized and tested for use in the imaging and treatment of colon cancer. Further, libraries of molecules can be screened for potential anticancer agents by assessing the ability of the molecule to bind to the CSGs identified herein. Molecules identified in the library as being capable of binding to CSG are key candidates for further evaluation for use in the treatment of colon cancer. In a preferred embodiment, these molecules will downregulate expression and/or activity of CSG in cells.
Adoptive Immunotherapy and Vaccines
Adoptive immunotherapy of cancer refers to a therapeutic approach in which immune cells with an antitumor reactivity are administered to a tumor-bearing host, with the aim that the cells mediate either directly or indirectly, the regression of an established tumor. Transfusion of lymphocytes, particularly T lymphocytes, falls into this category and investigators at the National Cancer Institute (NCI) have used autologous reinfusion of peripheral blood lymphocytes or tumor-infiltrating lymphocytes (TIL), T cell cultures from biopsies of subcutaneous lymph nodules, to treat several human cancers (Rosenberg, S. A., U.S. Pat. No. 4,690,914, issued Sep. 1, 1987; Rosenberg, S. A., et al., 1988, N. England J. Med. 319:1676-1680).
The present invention relates to compositions and methods of adoptive immunotherapy for the prevention and/or treatment of primary and metastatic colon cancer in humans using macrophages sensitized to the antigenic CSG molecules, with or without non-covalent complexes of heat shock protein (hsp). Antigenicity or immunogenicity of the CSG is readily confirmed by the ability of the CSG protein or a fragment thereof to raise antibodies or educate naive effector cells, which in turn lyse target cells expressing the antigen (or epitope).
Cancer cells are, by definition, abnormal and contain proteins which should be recognized by the immune system as foreign since they are not present in normal tissues. However, the immune system often seems to ignore this abnormality and fails to attack tumors. The foreign CSG proteins that are produced by the cancer cells can be used to reveal their presence. The CSG is broken into short fragments, called tumor antigens, which are displayed on the surface of the cell. These tumor antigens are held or presented on the cell surface by molecules called MHC, of which there are two types: class I and II. Tumor antigens in association with MHC class I molecules are recognized by cytotoxic T cells while antigen-MHC class II complexes are recognized by a second subset of T cells called helper cells. These cells secrete cytokines which slow or stop tumor growth and help another type of white blood cell, B cells, to make antibodies against the tumor cells.
In adoptive immunotherapy, T cells or other antigen presenting cells (APCs) are stimulated outside the body (ex vivo), using the tumor specific CSG antigen. The stimulated cells are then reinfused into the patient where they attack the cancerous cells. Research has shown that using both cytotoxic and helper T cells is far more effective than using either subset alone. Additionally, the CSG antigen may be complexed with heat shock proteins to stimulate the APCs as described in U.S. Pat. No. 5,985,270.
The APCs can be selected from among those antigen presenting cells known in the art including, but not limited to, macrophages, dendritic cells, B lymphocytes, and a combination thereof, and are preferably macrophages. In a preferred use, wherein cells are autologous to the individual, autologous immune cells such as lymphocytes, macrophages or other APCs are used to circumvent the issue of whom to select as the donor of the immune cells for adoptive transfer. Another problem circumvented by use of autologous immune cells is graft versus host disease which can be fatal if unsuccessfully treated.
In adoptive immunotherapy with gene therapy, DNA of the CSG can be introduced into effector cells similarly as in conventional gene therapy. This can enhance the cytotoxicity of the effector cells to tumor cells as they have been manipulated to produce the antigenic protein resulting in improvement of the adoptive immunotherapy.
CSG antigens of this invention are also useful as components of colon cancer vaccines. The vaccine comprises an immunogenically stimulatory amount of a CSG antigen. Immunogenically stimulatory amount refers to that amount of antigen that is able to invoke the desired immune response in the recipient for the amelioration, or treatment of colon cancer. Effective amounts may be determined empirically by standard procedures well known to those skilled in the art.
The CSG antigen may be provided in any one of a number of vaccine formulations which are designed to induce the desired type of immune response, e.g., antibody and/or cell mediated. Such formulations are known in the art and include, but are not limited to, formulations such as those described in U.S. Pat. No. 5,585,103. Vaccine formulations of the present invention used to stimulate immune responses can also include pharmaceutically acceptable adjuvants.
Vectors, Host Cells, Expression
The present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
Host cells can be genetically engineered to incorporate polynucleotides and express polypeptides of the present invention. For instance, polynucleotides may be introduced into host cells using well known techniques of infection, transduction, transfection, transvection and transformation. The polynucleotides may be introduced alone or with other polynucleotides. Such other polynucleotides may be introduced independently, co-introduced or introduced joined to the polynucleotides of the invention.
Thus, for instance, polynucleotides of the invention may be transfected into host cells with another, separate, polynucleotide encoding a selectable marker, using standard techniques for co-transfection and selection in, for instance, mammalian cells. In this case the polynucleotides generally will be stably incorporated into the host cell genome.
Alternatively, the polynucleotides may be joined to a vector containing a selectable marker for propagation in a host. The vector construct may be introduced into host cells by the aforementioned techniques. Generally, a plasmid vector is introduced as DNA in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. Electroporation also may be used to introduce polynucleotides into a host. If the vector is a virus, it may be packaged in vitro or introduced into a packaging cell and the packaged virus may be transduced into cells. A wide variety of techniques suitable for making polynucleotides and for introducing polynucleotides into cells in accordance with this aspect of the invention are well known and routine to those of skill in the art Such techniques are reviewed at length in Sambrook et al. cited above, which is illustrative of the many laboratory manuals that detail these techniques. In accordance with this aspect of the invention the vector may be, for example, a plasmid vector, a single- or double-stranded phage vector, a single- or double-stranded RNA or DNA viral vector. Such vectors may be introduced into cells as polynucleotides, preferably DNA, by well known techniques for introducing DNA and RNA into cells. The vectors, in the case of phage and viral vectors also may be and preferably are introduced into cells as packaged or encapsidated virus by well known techniques for infection and transduction. Viral vectors may be replication competent or replication defective. In the latter case viral propagation generally will occur only in complementing host cells.
Preferred among vectors, in certain respects, are those for expression of polynucleotides and polypeptides of the present invention. Generally, such vectors comprise cis-acting control regions effective for expression in a host operatively linked to the polynucleotide to be expressed. Appropriate trans-acting factors either are supplied by the host, supplied by a complementing vector or supplied by the vector itself upon introduction into the host.
In certain preferred embodiments in this regard, the vectors provide for specific expression. Such specific expression may be inducible expression or expression only in certain types of cells or both inducible and cell-specific. Particularly preferred among inducible vectors are vectors that can be induced for expression by environmental factors that are easy to manipulate, such as temperature and nutrient additives. A variety of vectors suitable to this aspect of the invention, including constitutive and inducible expression vectors for use in prokaryotic and eukaryotic hosts, are well known and employed routinely by those of skill in the art.
The engineered host cells can be cultured in conventional nutrient media, which may be modified as appropriate for, inter alia, activating promoters, selecting transformants or amplifying genes. Culture conditions, such as temperature, pH and the like, previously used with the host cell selected for expression generally will be suitable for expression of polypeptides of the present invention as will be apparent to those of skill in the art.
A great variety of expression vectors can be used to express a polypeptide of the invention. Such vectors include chromosomal, episomal and virus-derived vectors e.g., vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids, all may be used for expression in accordance with this aspect of the present invention. Generally, any vector suitable to maintain, propagate or express polynucleotides to express a polypeptide in a host may be used for expression in this regard.
The appropriate DNA sequence may be inserted into the vector by any of a variety of well-known and routine techniques. In general, a DNA sequence for expression is joined to an expression vector by cleaving the DNA sequence and the expression vector with one or more restriction endonucleases and then joining the restriction fragments together using T4 DNA ligase. Procedures for restriction and ligation that can be used to this end are well known and routine to those of skill. Suitable procedures in this regard, and for constructing expression vectors using alternative techniques, which also are well known and routine to those skill, are set forth in great detail in Sambrook et al. cited elsewhere herein.
The DNA sequence in the expression vector is operatively linked to appropriate expression control sequence(s), including, for instance, a promoter to direct mRNA transcription. Representatives of such promoters include the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name just a few of the well-known promoters. It will be understood that numerous promoters not mentioned are suitable for use in this aspect of the invention are well known and readily may be employed by those of skill in the manner illustrated by the discussion and the examples herein.
In general, expression constructs will contain sites for transcription initiation and termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs will include a translation initiating AUG at the beginning and a termination codon appropriately positioned at the end of the polypeptide to be translated.
In addition, the constructs may contain control regions that regulate as well as engender expression. Generally, in accordance with many commonly practiced procedures, such regions will operate by controlling transcription, such as repressor binding sites and enhancers, among others.
Vectors for propagation and expression generally will include selectable markers. Such markers also may be suitable for amplification or the vectors may contain additional markers for this purpose. In this regard, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells. Preferred markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, and tetracycline or ampicillin resistance genes for culturing E. coli and other bacteria.
The vector containing the appropriate DNA sequence as described elsewhere herein, as well as an appropriate promoter, and other appropriate control sequences, may be introduced into an appropriate host using a variety of well known techniques suitable to expression therein of a desired polypeptide. Representative examples of appropriate hosts include bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes melanoma cells; and plant cells. Hosts for a great variety of expression constructs are well known, and those of skill will be enabled by the present disclosure readily to select a host for expressing a polypeptide in accordance with this aspect of the present invention.
More particularly, the present invention also includes recombinant constructs, such as expression constructs, comprising one or more of the sequences described above. The constructs comprise a vector, such as a plasmid or viral vector, into which such a sequence of the invention has been inserted. The sequence may be inserted in a forward or reverse orientation. In certain preferred embodiments in this regard, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and there are many commercially available vectors suitable for use in the present invention.
The following vectors, which are commercially available, are provided by way of example. Among vectors preferred for use in bacteria are pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Among preferred eukaryotic vectors are PWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, PBPV, pMSG and PSVL available from Pharmacia. These vectors are listed solely by way of illustration of the many commercially available and well known vectors that are available to those of skill in the art for use in accordance with this aspect of the present invention. It will be appreciated that any other plasmid or vector suitable for, for example, introduction, maintenance, propagation or expression of a polynucleotide or polypeptide of the invention in a host may be used in this aspect of the invention.
Promoter regions can be selected from any desired gene using vectors that contain a reporter transcription unit lacking a promoter region, such as a chloramphenicol acetyl transferase ("cat") transcription unit, downstream of restriction site or sites for introducing a candidate promoter fragment; i.e., a fragment that may contain a promoter. As is well known, introduction into the vector of a promoter-containing fragment at the restriction site upstream of the cat gene engenders production of CAT activity, which can be detected by standard CAT assays. Vectors suitable to this end are well known and readily available. Two such vectors are pKK232-8 and pCM7. Thus, promoters for expression of polynucleotides of the present invention include not only well known and readily available promoters, but also promoters that readily may be obtained by the foregoing technique, using a reporter gene.
Among known bacterial promoters suitable for expression of polynucleotides and polypeptides in accordance with the present invention are the E. coli lacI and lacZ and promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR, PL promoters and the trp promoter. Among known eukaryotic promoters suitable in this regard are the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus ("RSV"), and metallothionein promoters, such as the mouse metallothionein-I promoter.
Selection of appropriate vectors and promoters for expression in a host cell is a well known procedure and the requisite techniques for expression vector construction, introduction of the vector into the host and expression in the host are routine skills in the art.
The present invention also relates to host cells containing the above-described constructs discussed above. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.
Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al. BASIC METHODS IN MOLECULAR BIOLOGY, (1986).
Constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).
Generally, recombinant expression vectors will include origins of replication, a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence, and a selectable marker to permit isolation of vector containing cells after exposure to the vector. Among suitable promoters are those derived from the genes that encode glycolytic enzymes such as 3-phosphoglycerate kinase ("PGK"), a-factor, acid phosphatase, and heat shock proteins, among others. Selectable markers include the ampicillin resistance gene of E. coli and the trp1 gene of S. cerevisiae.
Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type. Examples of enhancers include the SV40 enhancer, which is located on the late side of the replication origin at bp 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
Polynucleotides of the invention, encoding the heterologous structural sequence of a polypeptide of the invention generally will be inserted into the vector using standard techniques so that it is operably linked to the promoter for expression. The polynucleotide will be positioned so that the transcription start site is located appropriately 5' to a ribosome binding site. The ribosome binding site will be 5' to the AUG that initiates translation of the polypeptide to be expressed. Generally, there will be no other open reading frames that begin with an initiation codon, usually AUG, and lie between the ribosome binding site and the initiating AUG. Also, generally, there will be a translation stop codon at the end of the polypeptide and there will be a polyadenylation signal and a transcription termination signal appropriately disposed at the 3' end of the transcribed region.
For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the expressed polypeptide. The signals may be endogenous to the polypeptide or they may be heterologous signals.
The polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals but also additional heterologous functional regions. Thus, for instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification or during subsequent handling and storage. Also, region also may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art.
Suitable prokaryotic hosts for propagation, maintenance or expression of polynucleotides and polypeptides in accordance with the invention include Escherichia coli, Bacillus subtilis and Salmonella typhimurium. Various species of Pseudomonas, Streptomyces, and Staphylococcus are suitable hosts in this regard. Moreover, many other hosts also known to those of skill may be employed in this regard.
As a representative but non-limiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322. Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis., USA). These pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed.
Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, where the selected promoter is inducible it is induced by appropriate means (e.g., temperature shift or exposure to chemical inducer) and cells are cultured for an additional period.
Cells typically then are harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification. Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well know to those skilled in the art.
Various mammalian cell culture systems can be employed for expression, as well. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblast, described in Gluzman et al., Cell 23: 175 (1981). Other cell lines capable of expressing a compatible vector include for example, the C127, 3T3, CHO, HeLa, human kidney 293 and BHK cell lines.
Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking non-transcribed sequences that are necessary for expression. In certain preferred embodiments in this regard DNA sequences derived from the SV40 splice sites, and the SV40 polyadenylation sites are used for required non-transcribed genetic elements of these types.
The CSG polypeptide can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification. Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification.
Polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
CSG polynucleotides and polypeptides may be used in accordance with the present invention for a variety of applications, particularly those that make use of the chemical and biological properties of CSGs. Additional applications relate to diagnosis and to treatment of disorders of cells, tissues and organisms. These aspects of the invention are illustrated further by the following discussion.
This invention is also related to the use of the CSG polynucleotides to detect complementary polynucleotides such as, for example, as a diagnostic reagent. Detection of a mutated form of CSG associated with a dysfunction will provide a diagnostic tool that can add or define a diagnosis of a disease or susceptibility to a disease which results from under-expression, over-expression or altered expression of CSG, such as, for example, a susceptibility to inherited colon cancer.
Individuals carrying mutations in the CSGs may be detected at the DNA level by a variety of techniques. Nucleic acids for diagnosis may be obtained from a patient's cells, such as from blood, urine, saliva, tissue biopsy and autopsy material. The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR prior to analysis. PCR (Saiki et al., Nature, 324: 163-166 (1986)). RNA or cDNA may also be used in the same ways. As an example, PCR primers complementary to the CSG can be used to identify and analyze CSG expression and mutations. For example, deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to radiolabeled CSG RNA or alternatively, radiolabeled CSG antisense DNA sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures.
Sequence differences between a reference gene and genes having mutations also may be revealed by direct DNA sequencing. In addition, cloned DNA segments may be employed as probes to detect specific DNA segments. The sensitivity of such methods can be greatly enhanced by appropriate use of PCR or another amplification method. For example, a sequencing primer is used with double-stranded PCR product or a single-stranded template molecule generated by a modified PCR. The sequence determination is performed by conventional procedures with radiolabeled nucleotide or by automatic sequencing procedures with fluorescent-tags.
Genetic testing based on DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels, with or without denaturing agents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis. DNA fragments of different sequences may be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers et al., Science, 230: 1242 (1985)).
Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase and S1 protection or the chemical cleavage method (e.g., Cotton et al., Proc. Natl. Acad. Sci., USA, 85: 4397-4401 (1985)).
Thus, the detection of a specific DNA sequence may be achieved by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes, (e.g., restriction fragment length polymorphisms ("RFLP") and Southern blotting of genomic DNA.
In addition to more conventional gel-electrophoresis and DNA sequencing, mutations also can be detected by in situ analysis.
The sequences of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome. Moreover, there is a current need for identifying particular sites on the chromosome. Few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available for marking chromosomal location. The mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.
In certain preferred embodiments in this regard, the cDNA herein disclosed is used to clone genomic DNA of a CSG. This can be accomplished using a variety of well known techniques and libraries, which generally are available commercially. The genomic DNA is used for in situ chromosome mapping using well known techniques for this purpose. Typically, in accordance with routine procedures for chromosome mapping, some trial and error may be necessary to identify a genomic probe that gives a good in situ hybridization signal.
In some cases, in addition, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3' untranslated region of the gene is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment.
PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome. Using the present invention with the same oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner. Other mapping strategies that can similarly be used to map to its chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries.
Fluorescence in situ hybridization ("FISH") of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step. This technique can be used with cDNA as short as 50 or 60. For a review of this technique, see Verma et al., HUMAN CHROMOSOMES: A MANUAL OF BASIC TECHNIQUES, Pergamon Press, New York (1988).
Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in V. McKusick, MENDELIAN INHERITANCE IN MAN, available on line through Johns Hopkins University, Welch Medical Library. The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes).
Next, it is necessary to determine the differences in the cDNA or genomic sequence between affected and unaffected individuals. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.
With current resolution of physical mapping and genetic mapping techniques, a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes. (This assumes 1 megabase mapping resolution and one gene per 20 kb).
The present invention also relates to a diagnostic assays such as quantitative and diagnostic assays for detecting levels of CSG in cells and tissues, and biological fluids such, for example, as blood and urine, including determination of normal and abnormal levels. Thus, for instance, a diagnostic assay in accordance with the invention for detecting over-expression or under-expression of CSG compared to normal control tissue samples may be used to detect the presence of neoplasia, for example. Assay techniques that can be used to determine levels of a protein, such as CSG of the present invention, in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays. Among these ELISAs frequently are preferred. An ELISA assay initially comprises preparing an antibody specific to a CSG, preferably a monoclonal antibody. In addition a reporter antibody generally is prepared which binds to the monoclonal antibody. The reporter antibody is attached a detectable reagent such as radioactive, fluorescent or enzymatic reagent, for example horseradish peroxidase enzyme.
To carry out an ELISA a sample is removed from a host and incubated on a solid support, e.g. a polystyrene dish, that binds the proteins in the sample. Any free protein binding sites on the dish are then covered by incubating with a non-specific protein such as bovine serum albumin. Next, the monoclonal antibody is incubated in the dish during which time the monoclonal antibodies attach to any CSG attached to the polystyrene dish. Unbound monoclonal antibody is washed out with buffer. The reporter antibody linked to horseradish peroxidase is placed in the dish resulting in binding of the reporter antibody to any monoclonal antibody bound to CSG. Unattached reporter antibody is then washed out. Reagents for peroxidase activity, including a calorimetric substrate are then added to the dish. Immobilized peroxidase, linked to CSG through the primary and secondary antibodies, produces a colored reaction product. The amount of color developed in a given time period indicates the amount of CSG protein present in the sample. Quantitative results typically are obtained by reference to a standard curve.
A competition assay may be employed wherein antibodies specific to CSG attached to a solid support and labeled CSG and a sample derived from the host are passed over the solid support and the amount of label detected attached to the solid support can be correlated to a quantity of CSG in the sample.
The polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an immunogen to produce antibodies thereto. These antibodies can be, for example, polyclonal or monoclonal antibodies. The present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.
Antibodies generated against the polypeptides corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides. Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide.
For preparation of monoclonal antibodies, techniques which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler, G. and Milstein, C., Nature 256: 495-497 (1975), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., Immunology Today 4: 72 (1983) and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., pg. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).
Techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce single chain antibodies to immunogenic polypeptide products of this invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized antibodies to immunogenic polypeptide products of this invention.
The above-described antibodies may be employed to isolate or to identify clones expressing the polypeptide or purify the polypeptide of the present invention by attachment of the antibody to a solid support for isolation and/or purification by affinity chromatography.
CSG Binding Molecules and Assays
This invention also provides a method for identification of molecules, such as receptor molecules, that bind CSG. Genes encoding proteins that bind CSG, such as receptor proteins, can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting. Such methods are described in many laboratory manuals such as, for instance, Coligan et al., Current Protocols in Immunology 1(2): Chapter 5 (1991).
For instance, expression cloning may be employed for this purpose. To this end polyadenylated RNA is prepared from a cell responsive to CSG, a cDNA library is created from this RNA, the library is divided into pools and the pools are transfected individually into cells that are not responsive to CSG. The transfected cells then are exposed to labeled CSG. (CSG can be labeled by a variety of well-known techniques including standard methods of radio-iodination or inclusion of a recognition site for a site-specific protein kinase.) Following exposure, the cells are fixed and binding of cytostatin is determined. These procedures conveniently are carried out on glass slides.
Pools are identified of cDNA that produced CSG-binding cells. Sub-pools are prepared from these positives, transfected into host cells and screened as described above. Using an iterative sub-pooling and re-screening process, one or more single clones that encode the putative binding molecule, such as a receptor molecule, can be isolated.
Alternatively a labeled ligand can be photoaffinity linked to a cell extract, such as a membrane or a membrane extract, prepared from cells that express a molecule that it binds, such as a receptor molecule. Cross-linked material is resolved by polyacrylamide gel electrophoresis ("PAGE") and exposed to X-ray film. The labeled complex containing the ligand-receptor can be excised, resolved into peptide fragments, and subjected to protein microsequencing. The amino acid sequence obtained from microsequencing can be used to design unique or degenerate oligonucleotide probes to screen cDNA libraries to identify genes encoding the putative receptor molecule.
Polypeptides of the invention also can be used to assess CSG binding capacity of CSG binding molecules, such as receptor molecules, in cells or in cell-free preparations.
Agonists and Antagonists--Assays and Molecules
The invention also provides a method of screening compounds to identify those which enhance or block the action of CSG on cells, such as its interaction with CSG-binding molecules such as receptor molecules. An agonist is a compound which increases the natural biological functions of CSG or which functions in a manner similar to CSG, while antagonists decrease or eliminate such functions.
For example, a cellular compartment, such as a membrane or a preparation thereof, such as a membrane-preparation, may be prepared from a cell that expresses a molecule that binds CSG, such as a molecule of a signaling or regulatory pathway modulated by CSG. The preparation is incubated with labeled CSG in the absence or the presence of a candidate molecule which may be a CSG agonist or antagonist. The ability of the candidate molecule to bind the binding molecule is reflected in decreased binding of the labeled ligand. Molecules which bind gratuitously are most likely to be good antagonists. Molecules that bind well and elicit effects that are the same as or closely related to CSG are agonists. CSG-like effects of potential agonists and antagonists may by measured, for instance, by determining activity of a second messenger system following interaction of the candidate molecule with a cell or appropriate cell preparation, and comparing the effect with that of CSG or molecules that elicit the same effects as CSG. Second messenger systems that may be useful in this regard include, but are not limited to, AMP guanylate cyclase, ion channel or phosphoinositide hydrolysis second messenger systems.
Another example of an assay for CSG antagonists is a competitive assay that combines CSG and a potential antagonist with membrane-bound CSG receptor molecules or recombinant CSG receptor molecules under appropriate conditions for a competitive inhibition assay. CSG can be labeled, such as by radioactivity, such that the number of CSG molecules bound to a receptor molecule can be determined accurately to assess the effectiveness of the potential antagonist.
Potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to a polypeptide of the invention and thereby inhibit or extinguish its activity. Potential antagonists also may be small organic molecules, a peptide, a polypeptide such as a closely related protein or antibody that binds the same sites on a binding molecule, such as a receptor molecule, without inducing CSG-induced activities, thereby preventing the action of CSG by excluding CSG from binding.
Potential antagonists include a small molecule which binds to and occupies the binding site of the polypeptide thereby preventing binding to cellular binding molecules, such as receptor molecules, such that normal biological activity is prevented. Examples of small molecules include but are not limited to small organic molecules, peptides or peptide-like molecules.
Other potential antagonists include antisense molecules. Antisense technology can be used to control gene expression through antisense DNA or RNA or through triple-helix formation. Antisense techniques are discussed, for example, in --Okano, J. Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988). Triple helix formation is discussed in, for instance Lee et al., Nucleic Acids Research 6: 3073 (1979); Cooney et al., Science 241: 456 (1988); and Dervan et al., Science 251: 1360 (1991). The methods are based on binding of a polynucleotide to a complementary DNA or RNA. For example, the 5' coding portion of a polynucleotide that encodes the mature polypeptide of the present invention may be used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription thereby preventing transcription and the production of CSG. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into CSG polypeptide. The oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of CSG.
The antagonists may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as hereinafter described.
The invention also relates to compositions comprising the polynucleotide or the polypeptides discussed above or the agonists or antagonists. Thus, the polypeptides of the present invention may be employed in combination with a non-sterile or sterile carrier or carriers for use with cells, tissues or organisms, such as a pharmaceutical carrier suitable for administration to a subject. Such compositions comprise, for instance, a media additive or a therapeutically effective amount of a polypeptide of the invention and a pharmaceutically acceptable carrier or excipient. Such carriers may include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof. The formulation should suit the mode of administration.
The invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, reflecting approval by the agency of the manufacture, use or sale of the product for human administration.
Polypeptides and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
The pharmaceutical compositions may be administered in any effective, convenient manner including, for instance, administration by topical, oral, anal, vaginal, intravenous,
The pharmaceutical compositions may be administered in any effective, convenient manner including, for instance, administration by topical, oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes among others.
The pharmaceutical compositions generally are administered in an amount effective for treatment or prophylaxis of a specific indication or indications. In general, the compositions are administered in an amount of at least about 10 μg/kg body weight. In most cases they will be administered in an amount not in excess of about 8 μg/kg body weight per day. Preferably, in most cases, dose is from about 10 μg/kg to about 2 μg/kg body weight, daily. It will be appreciated that optimum dosage will be determined by standard methods for each treatment modality and indication, taking into account the indication, its severity, route of administration, complicating conditions and the like.
The CSG polynucleotides, polypeptides, agonists and antagonists that are polypeptides may be employed in accordance with the present invention by expression of such polypeptides in vivo, in treatment modalities often referred to as "gene therapy."
Thus, for example, cells from a patient may be engineered with a polynucleotide, such as a DNA or RNA, encoding a polypeptide ex vivo, and the engineered cells then can be provided to a patient to be treated with the polypeptide. For example, cells may be engineered ex vivo by the use of a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention. Such methods are well-known in the art and their use in the present invention will be apparent from the teachings herein.
Similarly, cells may be engineered in vivo for expression of a polypeptide in vivo by procedures known in the art. For example, a polynucleotide of the invention may be engineered for expression in a replication defective retroviral vector, as discussed above. The retroviral expression construct then may be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention such that the packaging cell now produces infectious viral particles containing the gene of interest. These producer cells may be administered to a patient for engineering cells in vivo and expression of the polypeptide in vivo. These and other methods for administering a polypeptide of the present invention by such method should be apparent to those skilled in the art from the teachings of the present invention.
Retroviruses from which the retroviral plasmid vectors herein above mentioned may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus. In one embodiment, the retroviral plasmid vector is derived from Moloney Murine Leukemia Virus.
Such vectors will include one or more promoters for expressing the polypeptide. Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller et al., Biotechniques 7: 980-990 (1989), or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, RNA polymerase III, and β-actin promoters). Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoters, and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.
The nucleic acid sequence encoding the polypeptide of the present invention will be placed under the control of a suitable promoter. Suitable promoters which may be employed include, but are not limited to, adenoviral promoters, such as the adenoviral major late promoter; or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters; the albumin promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs (including the modified retroviral LTRs herein above described); the beta-actin promoter; and human growth hormone promoters. The promoter also may be the native promoter which controls the gene encoding the polypeptide.
The retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, Y-2, Y-AM, PA12, T19-14×, VT-19-17-H2, YCRE, YCRIP, GP+E-86, GP+envAm12, and DAN cell lines as described in Miller, A., Human Gene Therapy 1: 5-14 (1990). The vector may be transduced into the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO4 precipitation. In one alternative, the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.
The producer cell line will generate infectious retroviral vector particles, which include the nucleic acid sequence(s) encoding the polypeptides. Such retroviral vector particles then may be employed to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express the nucleic acid sequence(s) encoding the polypeptide. Eukaryotic cells which may be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells.
The present invention is further described by the following example. The example is provided solely to illustrate the invention by reference to specific embodiments. This exemplification, while illustrating certain aspects of the invention, does not portray the limitations or circumscribe the scope of the disclosed invention.
All examples outlined here were carried out using standard techniques, which are well known and routine to those of skill in the art, except where otherwise described in detail. Routine molecular biology techniques of the following example can be carried out as described in standard laboratory manuals, such as Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).
Identification of CSGs
Identification of CSGs (Colon Specific Gene) was carried out by a systematic analysis of data in the LIFESEQ Gold database available from Incyte Pharmaceuticals, Palo Alto, Calif. using the data mining Cancer Leads Automatic Search Package referred to herein as CLASP.
CLASP performs the following steps. First, highly expressed organ specific genes are selected based on the abundance level of the corresponding EST in the targeted organ versus all the other organs. Next, the expression level of each highly expressed organ specific gene is analyzed in normal tissue, tumor tissue, and tissue libraries associated with tumor or disease. Candidates are selected based upon demonstration of components of ESTs as well as expression exclusively or more frequently in tumor tissue or tumor libraries.
Thus, CLASP allows the identification of highly expressed organ and cancer specific genes. A final manual in depth evaluation is then performed to finalize the gene selection.
Using the CLASP method, the following Incyte sequences were identified as CSGs.
TABLE-US-00001 SEQ ID NO: LSGold Gene ID 1 237623 2 234891 3 262167 4 246508 5 203279 6 983538 7 206344 8 222237 9 118593 10 337950 11 982786 12 398963 13 203640 14 88875 15 230552 16 407124 17 62662 18 230495 19 470880 20 898601 21 29586 22 370788
Relative Quantitation of Gene Expression
Real-Time quantitative PCR with fluorescent Taqman probes is a quantitation detection system utilizing the 5'-3' nuclease activity of Taq DNA polymerase. The method uses an internal fluorescent oligonucleotide probe (Taqman) labeled with a 5' reporter dye and a downstream, 3' quencher dye. During PCR, the 5'-3' nuclease activity of Taq DNA polymerase releases the reporter, whose fluorescence can then be detected by the laser detector of the Model 7700 Sequence Detection System (PE Applied Biosystems, Foster City, Calif., USA).
Amplification of an endogenous control is used to standardize the amount of sample RNA added to the reaction and normalize for Reverse Transcriptase (RT) efficiency. Either cyclophilin, glyceraldehyde-3-phosphate dehydrogenase (CAPDH) or 18S ribosomal RNA (rRNA) was used as this endogenous control. To calculate relative quantitation between all the samples studied, the target RNA levels for one sample was used as the basis for comparative results (calibrator). Quantitation relative to the "calibrator" can be obtained using the standard curve method or the comparative method (User Bulletin #2: ABI PRISM 7700 Sequence Detection System).
The tissue distribution and the level of the target gene were determined for each sample of normal and cancer tissue. Total RNA was extracted from normal tissues, cancer tissues and from cancers and the corresponding matched adjacent tissues. Subsequently, first strand cDNA was prepared with reverse transcriptase and the polymerase chain reaction was done using primers and Taqman probe specific to each target gene. The results were analyzed using the ABI PRISM 7700 Sequence Detector. The absolute numbers are relative levels of expression of the target gene in a particular tissue compared to the calibrator tissue.
The following primers were used for real-time quantitative PCR:
TABLE-US-00002 forward primer: TGGAAATAGATTCAGGGGTCAT (SEQ ID NO:23) reverse primer: CGGGTGTACCTCACTGACTTC (SEQ ID NO:24) Q-PCR probe: TGTCTTCCGAGAGAACCAGGCTCCG (SEQ ID NO:25)
The absolute numbers depicted in Table 1 are relative levels of expression of Gene ID 203279 (also referred to herein as Cln129 or SEQ ID NO:5) in 24 normal different tissues. All the values were compared to normal liver (calibrator). These RNA samples are commercially available pools, originated by pooling samples of a particular tissue from different individuals.
TABLE-US-00003 TABLE 1 Relative Levels of CSG Cln129 Expression in Pooled Samples TISSUE NORMAL Adrenal Gland 0 Bladder 0 Brain 0 Cervix 0 Colon 0.7 Endometrium 0.4 Esophagus 0 Heart 0 Kidney 3.7 Liver 1 Lung 0 Mammary Gland 0.2 Muscle 0 Ovary 0 Pancreas 0 Prostate 0 Rectum 23 Small Intestine 1.5 Spleen 0 Stomach 0.8 Testis 0.1 Thymus 0.4 Trachea 0 Uterus 0
The relative levels of expression in Table 1 show that Cln129 mRNA expression is detected at high levels in the pool of normal rectum (23), and at a lower levels in kidney (3.7). In contrast, Cln129 is expressed at very low levels in the other 22 normal tissue pools analyzed. Further, the level of expression in rectum is 6 fold higher compared to the expression in kidney. These results demonstrate that Cln129 mRNA expression is highly specific for rectum tissue.
The absolute numbers in Table 1 were obtained analyzing pools of samples of a particular tissue from different individuals. They can not be compared to the absolute numbers originated from RNA obtained from tissue samples of a single individual in Table 2.
The absolute numbers depicted in Table 2 are relative levels of expression of Cln129 in 21 pairs of matching samples. All the values are compared to normal liver (calibrator). A matching pair is formed by mRNA from the cancer sample for a particular tissue and mRNA from the normal adjacent sample for that same tissue from the same individual.
TABLE-US-00004 TABLE 2 Relative Levels of CSG Cln129 Expression in Individual Samples Sample ID Tissue CANCER NORMAL ClnAS98 Colon ascending (C)1 383 24 ClnCM67 Colon cecum (B)2 15 8 ClnCXGA Colon rectum (A)3 85 118 ClnMT38 Colon splenic flexture (D)4 33 18 ClnRC24 Colon rectum (D)5 77 29 ClnRC67 Colon rectum (B)6 0.9 15 ClnRS45 Colon rectosigmoid (C)7 161 25 ClnSG27 Colon sigmoid (C)8 48 13 ClnSG33 Colon sigmoid (C)9 190 100 ClnSG36 Colon sigmoid (B)10 186 93 ClnRC89 Colon rectum (D)11 0 28 Bld32XK Bladder 1 0 0 CvxKS52 Cervix 1 0 0 Endo8XA Endometrium 1 0 0.7 Kid106XD Kidney 1 0 6.7 Liv15XA Liver 1 1.7 3.2 Lng47XQ Lung 1 3.4 0 Mam59X Mammary Gland 1 1.3 0 Pro34B Prostate 1 0 0 SmInt 21XA Small Intestine 1 5.4 1.7 Utr85XU Uterus 1 0.9 0 0= Negative
Among 42 samples in Table 2 representing 11 different tissues significant expression is seen only in colon, kidney, and small intestine tissues. These results confirm the tissue specificity results obtained with normal samples shown in Table 1. Table 1 and Table 2 represent a combined total of 66 samples in 24 human tissue types. Only one small intestine sample, one lung sample, one liver sample, and one kidney sample showed expression of Cln129, out of a total of forty-two samples representing 22 different tissue types different than colon and rectum.
Comparisons of the level of mRNA expression in colon cancer samples and the normal adjacent tissue from the same individuals are shown in Table 2. Cln129 is expressed at higher levels in 8 of 11 (73%) cancer samples (colon 1, 2, 4, 5, 7, 8, 9, 10) compared to normal adjacent tissue.
Altogether, the high level of tissue specificity, plus the mRNA upregulation in 73% of the colon cancer matching samples tested indicate Cln129 to be a diagnostic marker for colon cancer.
251911DNAHomo sapien 1tttttttttt ttgcctgttt gttcataatg tttactgtac aaagaaacaa aacccaggaa 60tagtacaagt attgaacagt agcgagagtg gttgtgaaat aaaggaccac tttggaagac 120agttttattg gcttgctgtc ttcaccaaga aagacttgtg atttttgaaa acttctacct 180gaaatgtatt ttttctgctt tcccgaggaa gcggcactta cagtgttcct aggctttcct 240gtgacgtggg tgccagtctg gattcaaaat atccttgcat gcactgcagc tccttaggga 300gtcttttcct gcccttgagg cctgggcaga ctctcccctg acaccctccc gccctctccc 360acgacgcagc agaaataaag cacaacctca gaaagtctca ggcacgaaga actgtcctcg 420ggtggagcat gggaccttta ttcgttaaga catcaggctc cagatatgaa ctttcagcag 480aagcgcttgc cgggagcaaa gggacagaaa agctgagatg aacagtgcct ggcagcaatc 540acagccgggc aagggtgctc cgagcctcgc atcccccggc cgggggcagc tggaggtgcc 600tcagaaggtg cattctgctt cctgcagggg cttgaaacac caaggcactc cagggatcct 660ggagtcaaag cagcagcccc ggttgttgca ctccttgggg gtgacatggg ggtagccgca 720gtccaccctg tccttggctg gcacggcaca ctggtttgca gctgtcccag acaaagccct 780gtcagctgcc agagcccttg ctgggacagg cccacgtact tcctcagcag agctggagga 840cagcaaggcc aggaccagcc ccagcatgca gagcgctctg gcagccatga ccaccgtggg 900ctccgggacg c 9112322DNAHomo sapiensmisc_feature(244)..(244)n=a, c, g or t 2gacaagcaac aaacccttga tgattattca tcacttggat gagtgcccac acagtcaagc 60tttaaagaaa gtgtttgctg aaaataaaga aatccagaaa ttggcagagc agtttgtcct 120cctcaatctg gtttatgaaa caactgacaa acacctttct cctgatggcc agtatgtccc 180caggattatg tttgttgacc catctctgac agttagagcc gatatcactg gaagatattc 240aaancgtctc tatgcttacg aacctgcaga tacagctctg ttgcttgaca acatgaagaa 300agctctcaag ttgctgaaga ct 32234569DNAHomo sapiens 3atggataaat tcctcaacac atacactctc ccaagactaa accaggaaga agttgaatct 60ctgaatagac caataacagg ctctgatatt gtggcaataa tcaagagctt accaaccaaa 120aagagtccag gaccagatgg attcacagct gaattctacc agaggtacaa ggaggaactg 180gtaccattcc ctctgaaagt attacaatca atagaaaaag aggcaatcct ccctaactcg 240ttttatgagg ccaacatcat cctgatacca aagccgggca gagacacaac caaaaaagag 300aattttagac caatatcttt gatgaacatt gatgcaaaaa tcctcaataa aatactggca 360aaccgaatcc agcagcacat caaaaagctt atccaccatg atcaagtggg cttcatccct 420gggataacca aagacaaaaa ccacatgatt atctcaatag atgcagaaaa ggcctttgac 480aaaattcaac aacccttcat gctaaaaacc ctcaataaat tagatattga tgggacatat 540ctcaaaataa taagagctat ctatggcaaa gccacagcca atatcatact gaatgggcaa 600aaactggaag cattcccttt gaaaactggc acaagacagg gatgccctct ctcaccactc 660ctattcaaca tagttttgga agttctggcc agggcaatta ggcaggagaa ggaaataaag 720ggttttcaat taggaaaaga ggaagtcaaa ttgtccctgt ttgcaggtga catgattgta 780tacctagaaa accccattct ctcagcccaa aatctcctta agctgataag caacttcagc 840aaagtctcag gatacaaaat caatgtacaa aaatcacaag cattcctata caccaataac 900agagaaacag agagccaaat catgaatgaa ctcccattca caattgcttc aaagagaata 960aaatacctag gaatccaact tacaagggat gtgaaggacc tcttcaagga gaactacaaa 1020ccactgctca atgaaataaa agaggataca aacaaatgga agaacattcc atgctcatgg 1080ataggaagaa tcaatatcgt gaaaatggcc atactgccca agattatgct agatataaag 1140ggtattcaat taggaaaaga ggaagtcaaa ttgtccctgt ttgcagatga catgattgta 1200tatctagaaa accccattgt ctcagcccaa aatctcctta agctgataag caacttcagc 1260aaagtctcag gatacaaaat caatgtacaa aaatcacaag cattcttata caccaacaac 1320agacaaacag agagccaaat catgagtgaa ctcccattca caattgcttc aaagagaata 1380aaatacctag gaatccaact tacaagggac gtgaaggacc tcttcaagga gaactacaaa 1440ccactgctca aggaaataaa agaggataca aacaaatgga agaacatttc atgctcatgg 1500ataggaagaa tcaatatcgt gaaaatggcc atactgccca agagagaaat cacagggaga 1560tgtacagcaa tggggccatt taagagttct gtgttcatct tgattcttca ccttctagaa 1620ggggccctga gtaattcact cattcagctg aacaacaatg gctatgaagg cattgtcgtt 1680gcaatcgacc ccaatgtgcc agaagatgaa acactcattc aacaaataaa gggggagtac 1740acgtcacaag atgaggaagg gagagtcaga gagaaactct ctcttccccc gtcaaatata 1800catacacaca caccacacgc acaagctcgt gtgcacacac acacgcccat gcacacacgc 1860agacatacac gcacacacgc acgtcagaag gacatggtga cccaggcatc tctgtatctg 1920cttgaagcta caggaaagcg attttatttc aaaaatgttg ccattttgat tcctgaaaca 1980tggaagacaa aggctgacta tgtgagacca aaacttgaga cctacaaaaa tgctgatgtt 2040ctggttgctg agtctactcc tccaggtaat gatgaaccct acactgagca gatgggcaac 2100tgtggagaga agggtgaaag gatccacctc actcctgatt tcattgcagg aaaaaagtta 2160gctgaatatg gaccacaagg tagggcattt gtccatgagt gggctcatct acgatgggga 2220gtatttgacg agtacaataa tgatgagaaa ttctacttat ccaatggaag aatacaagca 2280gtaagatgtt cagcaggtat tactggtaca aatgtagtaa agaagtgtca gggaggcagc 2340tgttacacca aaagatgcac attcaataaa gtaacaggac tctatgaaaa aggatgtgag 2400tttgttctcc aatcccgcca gacggagaag gcttctataa tgtttgcaca acatgttgat 2460tctatagttg aattctgtac agaacaaaac cacaacaaag aagctccaaa caagcaaaat 2520caaaaatgca atctccgaag cacatgggaa gtgatccgtg attctgagga ctttaagaaa 2580accactccta tgacaacaca gccaccaaat cccaccttct cattgctgca gattggacaa 2640agaattgtgt gtttagtcct tgacaaatct ggaagcatgg cgactggtaa ccgcctcaat 2700cgactgaatc aagcaggcca gcttttcctg ctgcagacag ttgagctggg gtcctgggtt 2760gggatggtga catttgacag tgctgcccat gtacaaaatg aactcataca gataaacagt 2820ggcagtgaca gggacacact cgccaaaaga ttacctgcag cagcttcagg agggacgtcc 2880atctgcagcg ggcttcgatc ggcatttact gatatgtggc aacatttgcc tgttttccat 2940gacacacagc agttatgggg agtgcgacaa gaaaatccaa attgggcctc tctggcctgc 3000agcttagtga ttaggaagaa atatccaact gatggatctg aaattgtgct gctgacggat 3060ggggaagaca acactataag tgggtgcttt aacgaggtca aacaaagtgg tgccatcatc 3120cacacagtcg ctttggggcc ctctgcagct caagaactag aggagctgtc caaaatgaca 3180ggaggtttac agacatatgc ttcagatcaa gttcagaaca atggcctcat tgatgctttt 3240ggggcccttt catcaggaaa tggagctgtc tctcagcgct ccatccagct tgagagtaag 3300ggattaaccc tccagaacag ccagtggatg aatggcacag tgatcgtgga cagcaccgtg 3360ggaaaggaca ctttgtttct tatcacctgg acaatgcagc ctccccaaat ccttctctgg 3420gatcccagtg gacagaagca aggtggcttt gtagtggaca aaaacaccaa aatggcctac 3480ctccaaatcc caggcattgc taaggttggc acttggaaat acagtctgca agcaagctca 3540caaaccttga ccctgactgt cacgtcccgt gcgtccaatg ctaccctgcc tccaattaca 3600gtgacttcca aaacgaacaa ggacaccagc aaattcccca gccctctggt agtttatgca 3660aatattcgcc aaggagcctc cccaattctc agggccagtg tcacagccct gattgaatca 3720gtgaatggaa aaacagttac cttggaacta ctggataatg gagcaggtgc tgatgctact 3780aaggatgacg gtgtctactc aaggtatttc acaacttatg acacgaatgg tagatacagt 3840gtaaaagtgc gggctctggg aggagttaac gcagccagac ggagagtgat accccagcag 3900agtggagcac tgtacatacc tggctggatt gagaatgatg aaatacaatg gaatccacca 3960agacctgaaa ttaataagga tgatgttcaa cacaagcaag tgtgtttcag cagaacatcc 4020tcgggaggct catttgtggc ttctgatgtc ccaaatgctc ccatacctga tctcttccca 4080cctggccaaa tcaccgacct gaaggcggaa attcacgggg gcagtctcat taatctgact 4140tggacagctc ctggggatga ttatgaccat ggaacagctc acaagtatat cattcgaata 4200agtacaagta ttcttgatct cagagacaag ttcaatgaat ctcttcaagt gaatactact 4260gctctcatcc caaaggaagc caactctgag gaagtctttt tgtttaaacc agaaaacatt 4320acttttgaaa atggcacaga tcttttcatt gctattcagg ctgttgataa ggtcgatctg 4380aaatcagaaa tatccaacat tgcacgagta tctttgttta ttcctccaca gactccgcca 4440gagacaccta gtcctgatga aacgtctgct ccttgtccta atattcatat caacagcacc 4500attcctggca ttcacatttt aaaaattatg tggaagtgga taggagaact gcagctgtca 4560atagcctag 456943206DNAHomo sapiens 4ttcggctcga gtgtaaaact gccaaggaaa gtaattacct gtaggagttt gctgagcttg 60aagagtgaaa actgttgtga atgagcctga tcataaaacg gaccaggcca ttcattattc 120ctcaagtgtt aatatactga cttatgcagt attcaaacaa aaacattgca ctagatggtg 180caagaacagc gtaaaatgaa agccatcatt catcttactc ttcttgcgtc tcctttctgt 240aaacacagcc accaaccaag gcaactcagc tgatgctgta acaaccacag aaactgcgac 300tagtggtcct acagtagctg cagctgatac cactgaaact aatttgccct gaaactgcta 360gcaccacagc aaatacacct tctttcccaa cagctacttc acctgctccc cccataatta 420gtacacatag ttcctccaca attcctacac ctgctccccc cataattagt acacatagtt 480cctccacaat tcctatacct actgctgcag acagtgagtc aaccacaaat gtaaattcag 540ttagctacct ctgacataat caccgcttca tctccaaatg atggattaat tcacaatggt 600tccttctgaa acacaaagta acaatgaaat gtcccccacc acagaagaca atcaatcctc 660agtggcctcc cactgggcac cgctttattt ggatgaccat gcacgcctaa acagcacagt 720gtcccagcaa tccttgccaa agatgatccc cctgtgcaga taattcgtta ttgtttgtta 780agcttgctat aatacaagtt tttgcctgtg tttagaaggg tattactaca actcttctac 840atgtaagaaa ggaaaggtat tccctggaga agatttcagt gacagtatca gaaacatttg 900acccagaaga gaaacattcc atggcctatc aagacttgca tagtgaaatt actagcttgt 960ttaaagatgt atttggcaca tctgtttatg gacagactgt aattcttact gtaaggcaca 1020tctctgtcac caagattctg aaatgcgtgc ttgatgacaa gttttgttaa tgtaacaata 1080gtaacaattt tggcagaaac cacaagtgac aatgagaaga ctgtgactgg agaaaattaa 1140taaagcaatt tataagtagc tcaagcaact tttctaaact atgattggac cctgtcggtg 1200tggattgatt gagggctggg aaccaagact ggctggatga ctgcctcaat gggtttagca 1260tgcgatgtgc aaatgctgac ctgcaaaggc ctaacccaca gagccctttc tgcgttgctt 1320ccagtctcag agtgtcctga tgcctgcaac gcacagcaca agcgaatgct taataaagaa 1380gagtggtggg gtcccctgca gtgttgcgtt gcgtgcccgg tctaccagga agatgctaat 1440gggaactgcc aaaagtgtgc atttgggcta cagtggactc gactgtaagg acaaatttca 1500gctgatcctc acttatttgt gggcaccatc gctggcattg tcattctcag catgataatt 1560gcattgattg tcactagcaa gatcaaataa caaaagcgaa gcatattgaa gaacgagaac 1620ttgattgacg aagactttca aaatctaaaa ctgcggtcgc acaggcttca ccaatctatg 1680gagcataacg gagcgtcttc cctcaggtca ggattacggc ctccaagaga ccgcctagat 1740gcaaaaatcc cgtagtttca agacacagca gcatgccccc ggcctgacta ttagaatcca 1800tcagaatgtg gaacccgcca tggcccccaa ccatatgtac atatctatta ttctagcagt 1860gtttagacaa gactgcatgg agaagtgagc accacgtaaa gactctggcc tccgggagtt 1920tcttcttcca tctagacata ctgccagtcc tcatctgcaa tggcaacgtt gtgcaatgtc 1980ttgcaaacga catccacgct cacttgctaa aataagaatc tatgacatta acatgtagct 2040cgatgctatt agcgctgtgc tcagagaggt gggttttctt caatcagtaa caaagtactg 2100agacaatgct taggggttgg tttcttaatt cttttccctg gtagggcaac aagaccccat 2160ttccaaatct agaggaaagc ctccccagca ttgctttgct ccctgggcca aaccatgctt 2220cttgagttaa gttgacctaa cttcccctgg gacgacatac cgcatcaact gtggaggtcc 2280gagggggatg agaaagggat acccaccatc tttcataggg tcacaagcta cactctcgtg 2340acaagtcaga ataggggaca cctgcttcta tccctccaat ggaggagatt ctggccaaac 2400cccccttttt ttgaaaacca ggcccccaga gcttggcaac ctagcctcaa cccaagaaga 2460ctggaaagga gacatatctt ttcagctttt tcaggaggcg tgccttggga atccaggaac 2520gtttttgatg ctaattagaa ggcctggact ataataatgt ccatctatgg ggttttaatc 2580tacagttttt gaacatgcta ggaggcagaa cggggccaga gagtaaaaaa acatgacctg 2640gtagaaggaa gagaggcaaa ggaaactggg tggggaggat caattagaga ggaggcacct 2700gggatccacc ttcgttcctt aggtcccctc ctccatgcag caaaggagca cttctctaag 2760tcatgccctc ccgaagactg gctgggagaa ggtttaaaaa acaaaaaatc caggagtaaa 2820gagccttagg gtcagttttg aaaattggag acaaacttgt cttggcaaag ggtgccaaga 2880gcggagcttg ttgctcagga gtcccagccg tccagcctcg gggtgtaagg tctctgaggt 2940gtgccatggg ggcctcagcc ttctctggtg acccgaggct cagctgtggc caccaacaca 3000caaccacaca cacacaacca cacacacaaa tgggggcaac ccacatccac gtaaccaagc 3060tttaacacaa atgttattag tgtccctttt tatttctaat agccctgtcc tcttaaaagt 3120tattttattt gttattatta tttgttcttg actgttaatt gtgaatggta atgcaataaa 3180gtgcctttgt tagatggaaa aaaaaa 320652179DNAHomo sapiensmisc_feature(611)..(611)n=a, c, g or t 5gccttgcagc cgtttccctc tgcgattcat gtaagtgtga ctcgatttca gggaaaggga 60actcgcgtgg gctgaggaga ccggagtgga cgggctgggg aaggcaccgt gatgcccgca 120accccgtccc tgaaggtggt ccatgagctg cctgcctgta ccctctgtgc ggggccgctg 180gaggatgcgg tgaccattcc ctgtggacac accttctgcc ggctctgcct ccccgcgctc 240tcccagatgg gggcccaatc ctcgggcaag atcctgctct gcccgctctg ccaagaggag 300gagcaggcag agactcccat ggcccctgtg cccctgggcc cgctgggaga aacttactgc 360gaggagcacg gcgagaagat ctacttcttc tgcgagaacg atgccgagtt cctctgtgtg 420ttctgcaggg agggtcccac gcaccaggcg cacaccgtgg ggttcctgga cgaggccatt 480cagccctacc gggatcgtct caggagtcga ctggaagctc tgagcacgga gagagatgag 540attgaggatg taaagtgtca agaagaccag aagcttcaag tgctgctgac tcagatcgaa 600agcaagaagc ntcnggtgga gacagctttt gagaggctgg cagcaggagc tggagcagca 660gcgatgtctc ctgctggcca ggctgaggga gctggagcag cagatttgga agganaggga 720tgaatntatc acaaaggtct ctgaggaagt cacccggctt ggagcccagg tcaaggagct 780ggaggagaag tgtcagcagc cagcaagtga gcttctacaa gatgtcagag tcaaccagag 840caggtgtgag atgaagactt ttgtgagtcc tgaggccatt tctcctgacc ttgtcaagaa 900gatccgtgat ttccacagga aaatactcac cctcccagag atgatgagga tgttctcaga 960aaacttggcg catcatctgg aaatagattc aggggtcatc actctggacc ctcagaccgc 1020cagccggagc ctggttctct cggaagacag gaagtcagtg aggtacaccc ggcagaagaa 1080gaacctgcca gacagccccc tgcgcttcga cggcctcccg gcggttctgg gcttcccggg 1140cttctcctcc gggcgccacc gctggcaggt tgacctgcag ctgggcgacg gcggcggctg 1200cacggtgggg gtggccgggg agggggtgag gaggaaggga gagatgggac tcagcgccga 1260ggacggcgtc tgggccgtga tcatctcgca ccagcagtgc tgggccagca cctccccggg 1320caccgacctg ccgctgagcg agatcccgcg cggcgtgaga gtcgccctgg actacgaggc 1380ggggcaggtg accctccaca acgcccagac ccaggagccc atcttcacct tcactgcctc 1440tttctccggc aaagtcttcc ctttctttgc cgtctggaaa aaaggttcct gccttacgat 1500gaaaggctga agtggggcgc gcgaagggcg gcgaagcgga gacggcggct ctccgggatc 1560cagctccgcc cctggccagt gtgcggcccg ggggctccct gtgcccgcgt gaggcgagag 1620aacaggggac ttgagtctcg aacagcggtt gtttttactt tatttatctt aggccctcag 1680ctccctgacg tcctgagcct ccctgtgacg ctctggcctt ctctgcacct cagagtgcag 1740aaccacagac ggcttcggct gtgcctaggg caacagccaa cctaggagcc agcgggcttt 1800cggggaaaaa aaagaaaaag acatctaaaa taaaatgttt aaactgtttc aaaataatta 1860tcttgggaaa aatcagggtt ttgctggact tgcactaatt tgtacagtta acttcgtact 1920ttgacacaca cctgaagatg cctccacctt tgtagggctt agggcctttt tatcagccct 1980gggtggaccc cagggcccct tcctttccct tcccttctgg tcatttctct ggacttgtag 2040agaatgtcct aagaaagtgt gactcacaga cctctggatt ccatgtgtcc aattagcgct 2100gatgggactg gagaaaggct taaatccaat gggatcttgc ctgtgttggc aatttagggc 2160cgagatggct cgagggagt 217961627DNAHomo sapiens 6ttttattttc tagagtgata tatatttttt ggtctttttc tttttttttc ttccaaaaca 60aacaattaga gctttaggcc cctcgccctc cccacaccca ccgcagaacc ctcccatata 120atcgacaact gaaaacaagc gagacaatca cccccaaaga gatcacgaaa cacgagcaca 180agtttcacag acagccaccg acaaagcaaa aaaacttgct actaggaatg tccgccttgc 240atgatcatgt agaagcagga gcaagagtct acaaattgaa tggggacctg attaagtatg 300gggtagcagg gggatggtac ggaatcagaa gagtaaagct tccatgctga tgcgttaggt 360gccattttgc ccctttcctg ttgcacggcg ggtactgttt tcccagaagc gcgcgcacgc 420acctggccac gcagatctgc agtcctaggc cctgtgtagt caggatgtcc atagcccggt 480ccctggggcg ggtctccttt ggcgctgggg ctagagccgc caagcccggg gcttctctgc 540gtgggtcgag aagccgacgg gattcggagg aacgctgcag agcgttgtcg cactggggcc 600gttgcatcct ccctgtccca tgtaccactt gtacccggaa gggagtcatt gggaatcgag 660tgcgcaaata aattctcatt cggactctcc tggcctggct ttcctgtcta cagtggggtt 720gacactagcg gtggaacgga aggtggaggg atttttctac aaggggcggc ttgacttgcg 780ggtgcaaggt ggatacgacc gaagagagtt gatttcagag ctagggaggg tgcggaagaa 840tgcagtgccg gtcgaagagc aagagaagct acagtctgtc aagtggtgca cagatgaaca 900ggaggacaac attgtcaagg ctcatacgac ccacagtgtg accttatttt gttggaagga 960tgagggaaac atcatgctgg taaatataac atttcgtgca acaataatgt atataatggt 1020gggaggtggg gagtagctcc acctaagata ccttcataaa accacgtgct gccttttctt 1080gtactttcta gcccaccggc ttgggggcta ggtttgctcc atcttcccca tggcccttgg 1140cctgagaata gttggccact ccatgggaat ggtatggcca tgctgcagcc tttgggctgc 1200aactcctcac tcaggagtct gcctctagac atctccctgg tgggtatttg cattaggggt 1260agaacccggg cttgcctgac agtctgaggg ctgttttgcc caatttggtg tgcgatggtc 1320tgcaactggt agtgtcacct cacttgactg aatggtggtt gtgagctcac cccattactg 1380tgtgtgaatg tctgctgagc tgtgtagagt tggagtgtcc ctgggtgact tttgggtggg 1440tgtagagaag aaacaggcaa gctggaagtg aggggctagg acttcccaga aaaattacag 1500ggcatactag gagcttgact ggggtctctc tttccttgtg gcccatcaca ttcttaggaa 1560ccaactattt ctatcttcta aatcaacaaa actttctcct gacacctaga gacctgagca 1620agccatg 16277929DNAHomo sapiens 7catgtatgca ataaaaaata aaagatacat acacaaaatt ctttaaatgt cccacacaca 60agacaaatac gtgttcaaat acatcagtct ctgaagcctc tgcaccactc tacacgctgc 120tccttctgac tagtaatgcc ctcctgcccc tcctgtccac gtgtcaaact cccaatcacc 180ctttaaaacc agattgaatt attttgcttc tgtgaagctt tccctgacta tccccgggat 240agaataatgt ttccactagt gttttgtcat ttactcgcta taataagaat acgaaagaac 300atgtattttt gaaaagtatc tgtgatctct aatgagcttg taaacatctt gaggaataga 360gactaagttt tgcttctttg ttcccccaaa gagaacttta ttaataacat ttaccatctc 420tttagagaga gggtttttcc catctctgtg agaaagctcc agaatctaca accaggaata 480agtgttaatg ggatagaacc aatgtagaga acagcatatg atatgtgaaa tgtactttat 540tattaatacg aattcagtgg gctcacagaa tgaacctttt tgccaaactg gggggaaagc 600attttctgta aaggtatctt tagaaaaata tgtataattt gaaaaatggt tatccaaatt 660taacatttgt catataaaag gctcataaaa cgtgtgtggc tgtgtttctc aaaattgtgg 720ggtcaattgg tcacattatg cctagacatt ctggttttgt tgcttggggt taataatggt 780tgtggtctta tacagaaaag gaaatctgga catcttgccc ctgttattaa tacacctgtc 840attactaata aaagtggttt gttgatatgc taaataggtt gaaaaagctg tcactttgca 900tgaaattaac tagggaatac ttctttata 92982303DNAHomo sapiens 8gagaggaagc agcatcagga caccttacca ccactgccgc tgcctcagca tccaccccgc 60agcccacgtg tggcaaaccg gggaaggggt ggagtgaacg gccggagacc acgtggagaa 120aggggccgct ttggcccttc catctgggtg ccgggagccc ctaggccctc cggccatggc 180cgacagcggc gatgctggca gctccggccc ctggtggaaa tcgctcacca acagcagaaa 240gaaaagcaag gaagccgcag tgggggtgcc gcctcccgcc cagcccgctc ccggggagcc 300cacgccacct gcgccgccca gcccggactg gaccagcagc tcccgggaga accagcaccc 360ccaatctcct cgggggcgcc ggcgagcccc ccaaaccaga caagttatac ggggacaaat 420ccggcagcag ccgccgcaat ttgaagatct cgcgctccgg ccgctttaag gagaagagga 480aagtgcgcgc cacgctgctc ccggaggcgg gcaggtcctc ggaggaggca ggctttcctg 540gtgaccccca cgaggacaag cagtagcccc aatagcctgc gcgctccagg actgcctacc 600cagcactacc ccaaaccccc agttccaaac ccgagacttc aggcccgccc ccttacgcgt 660tgtctcattc caccaaattc agaatattta cacaatgcct tcatgattaa atttttctgg 720aacttgaagt gtcaattggg ttctcaagat ttcatgacgc caaggatgcc ttgaatattt 780atttgtggta agagaagata
cctgccgcgg agtagggtgg cataattatt ttttttctac 840agtgcaaggg ttttaatagt ccacactaaa ataggctgta cacttttgta gtttaacatc 900tcaaagcaat cctgccttat gtttaaaatg cttctactta agaatgcttc tgtcctcccc 960gcactccgtt cacttacagg tataagtcta cccctagaag tgcatttctc acggcaatta 1020aaaactagca ctgtgatttg ctttcctaca gagtcctgaa ataactagcc accttccttg 1080catttgatga ggctactaga gttccaagct cgagctcgtg actaggagca cagggggcca 1140gggcccacag aatacgcttt cttagaagaa aaaactaatt atgccaccct tcttccgcgg 1200caggtatcta tctcttacca caaataaata tttacaatgc atccttggga gtcatgaaat 1260attgagaacc caataagaca ctacaatttc cagaaaaata aaatcatgaa ggcattgctg 1320taaatattct gcaatttggt ggaatgagaa caacgcgtaa gggggcggac ctgaagtctc 1380ggttttggaa ctgggggttt agaggtagtg ctgggtaggc agtcctggag cgcgcaggct 1440attggggcta ctgcttgtcc tcgtgggggt caccaggaaa gcctgcctcc tccgaggacc 1500tgcccgcctc cgggagcagc gtggcgcgca ctttcctctt ctccttaaag cggccggagc 1560gcgagatctt caacattgcg gcggctgctg ccggatgtgt ccccgtataa cttgtctggt 1620ttggggggct cgccggcgcc cccgaggaga cttcggggtg ctggttctcc cgggagctgc 1680tggtccagtc cgggctgggc ggcgcaggtg gcgtgggctc cccgggagcg ggctgggcgg 1740gaggcggcac ccccactgcg gcttccttgc ttttctttct gctgttggtg agcgatttcc 1800accaggggcc cgagctgcca gcatcgccgc tgtcggccat ggccggaggg cctaggggct 1860cccggcaccc agatggaagg gccaaagcgg cccctttctc cacgtggtct ccggccgttc 1920actccacccc ttccccggct tgccacacgt ggggctgcgg ggtggatgct gaggcagcgg 1980cctgtgctgg gaggagggcc ctgggaacca agtgcatcct ctctacaggt gaacggtatt 2040aattaagtcc atggtcaaac aagtcacgaa atttccctcc aaagatttgc ccccatcgac 2100tttcgtccca ggaagccttt tcgatgagat acttaggaga attttatatc ccagttagga 2160agagaaggac aagcttatga tatttggttt tgggttcctt ttaaaattct ggcttttgac 2220caattctgcc ttgtgacttt caaagaagca tgtctagact taactttccc ttgaaaaacg 2280gcatcctaaa tcttcccttt act 230391769DNAHomo sapiensmisc_feature(878)..(948)n=a, c, g or t 9attctccagt cacttcctat agacttctgg cttcctgtca ggcatataac aagcttgaaa 60tttgtcactg gtttctaacg ctaagtaaaa agctgaacaa actcaaaagt caacaacttg 120ttaaaatccc tcagagatgg ctgggcactc catctctgag tggactcttg accccatcct 180cactcatgac gccatcctca acctgctgtg gcgctcatat cctccagtgg atcctgggac 240ctcccccagg tggagctggc caggcaggtg ctgtctgata ggtttgctgc ccattccaca 300tacacctgtg tcctcatgat gatgccattg tcataaggtg gagtcccttg gactgagaag 360tgaaccagcc actggcgtct cacttagact ctacccagtt acaaaaactt aaactctagt 420tgtgttttct gaggttgata ggagaggaag aaaacctttc acatgcctgt tttgaggctt 480ctcctctttt tgcctaactc tgcacaggaa ctaggggcag ggagcgcttt ctaaatttac 540taacatcaca cacattgctt ctcctaactt ggcatcattt ctccctttat gtaactgaca 600cacacctaag agttcctctc tgaccggttc tgtcctctta acaggtctca catccctctc 660tctgttcagg gagtcactga tttcaaacca ctttcagcat cttgccttag agcataatgt 720gatcactttg gaattcagag cagacctaaa ccttagcata atattaaaat gaaatactac 780ttcctagcaa attagataat tagatcttta ggaccaatga taagaattgt ccaccttatg 840gaaaagactt taaggtgttc ccccaaatgt ctttcacnnn nnnnnnnnnn nnnnnnnnnn 900nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnac tacagattga 960gtatcccaaa tccgaaaatc caaaaatcca aaatgtacca aaaatctgaa atgctcccaa 1020aatccaaaac ttttgagtgc caacataaca attaaaacaa aaatgctcac tggagcattt 1080cggatttggg attggatttt ggattttcag attagggatg ctcagctggg tgtcagatgc 1140ctgatacatt caattcatgg tttcttataa ccctactcca cgtctgggag atttatgtag 1200ttggaatttg tgttggcatt gtaagtgtta acagatttgt agagactccc cttttcaaat 1260tgtcatggag cactagtacc ttctcagtgc agaaattaat tttacaaaat ggaatggaac 1320aaataaaatt ggaacatacc tatgatggag gctgtcctgt ggccctcatg ctccccccag 1380aagggttagg cttcatagtg agggagtttg ggaaaccagg tggagatagc catgtacaca 1440gccctggaaa agggatgtgt ctagtccgaa tgaagcagga aggccggagt gggaagtaca 1500tgtgtcgtat catagttcat tttatgtggg aggatgttca gcagcgcggc agagtcatgg 1560ggtgggttcg tggtctcgct gacttcaaga atgaagccgc agaccttcac agcaagtgtt 1620accagctctt aaaggtggtg cggacccaaa gagtgagcag cagcaagatt tatggtgaag 1680accgaaagaa caaagcttcc acagtgtgga agggggacct gagcgggttg ccactgctgg 1740ctaggggcaa agttctccct gtggactga 1769102159DNAHomo sapiens 10cactagcaga gaagctgttg tccttccacc accagcaccg gaccacctgc tccaagacca 60gcctcctggg gggaccaggc acccggcctt cactggcacc cagggagccg tcctcagcag 120cgtcaacatg tcaaggccca gcagcagagc catttacttg caccggaagg agtactccca 180gaacctcacc tcagagccca ccctcctgca gcacagggtg gagcacttga tgacatgcaa 240gcaggggagt cagagagtcc aggggcccga ggatgccttg cagaagctgt tcgagatgga 300tgcacagggc cgggtgtgga gccaagactt gatcctgcag gtcagggacg gctggctgca 360gctgctggac attgagacca aggaggagct ggactcttac cgcctagaca gcatccaggc 420catgaatgtg gcgctcaaca catgctccta caactccatc ctgtccatca ccgtgcagga 480gccgggcctg ccaggcacta gcactctgct cttccagtgc caggaagtgg gggcagagcg 540actgaagacc agcctgcaga aggctctgga ggaagagctg gagcaaagac ctcgacttgg 600aggccttcag ccaggccagg acagatggag ggggcctgct atggaaaggc cgctccctat 660ggagcaggca cgctatctgg agccggggat ccctccagaa cagccccacc agaggaccct 720agagcacagc ctcccaccat ccccaaggcc cctgccacgc cacaccagtg cccgagaacc 780aagtgccttt actctgcctc ctccaaggcg gtcctcttcc cccgaggacc cagagaggga 840cgaggaagtg ctgaaccatg tcctaaggga cattgagctg ttcatgggaa agctggagaa 900ggcccaggca aagaccagca ggaagaagaa atttgggaaa gaagagaaca aggaccaggg 960aggtctcacc caggcacagt acagttgact gcttccagaa gatcaagcac agcttcaacc 1020tcctgggaag gctggccacc tggctgaagg agacaagtgc ccctgagctc gtacacatcc 1080tcttcaagtc cctgaacttc atcctggcca ggtgccctga ggctggccta gcagcccaag 1140tgatctcacc cctcctcacc cctaaagcta tcaacctgct acagtcctgt ctaagctcac 1200ctgagagtaa cctttggatg gggttgggcc cagcctggac cactagccgg gccgactgga 1260caggcgatga gcccctgccc taccaaccca cattctcaga tgactggcaa cttccagagc 1320cctccagcca agcaccctta ggataccagg accctgtttc ccttcgggcc tccagtcccc 1380aaacctgccc agccagtccc tgaaaatgca agtcttgtac gagtttgaag ctaggaatcc 1440cacgggaaac tgactgtggt ccaggtagag aagctggagg ttctggacca cagcaagcgg 1500tggtggctgg tgaagaatga ggcgggacgg agcggctaca ttccaagcaa catcctggag 1560cccctacagc cggggacccc tgggacccag ggccagtcac ccctctcggg ttccaatgct 1620tcgacttagc tcgaggcctg aagaggtcac agactggctg caggcagaga acttctccac 1680tgccacggtg aggacacttg ggtccctgac gggggagccc agctacttcg cattaagacc 1740tggggagcta ccaggatgct atgtccacca ggaggccccc acgaaatcct gtcccggctg 1800gaggctgtca gaaggatgct tggggataag cccttaggca ccagcttaga cacctccaag 1860aaccaggccc cgctgatgca agatggcaga tctgataccc attagagccc cgagaattcc 1920tcttctggat cccagtttgc agcaaacccc acacctccag cgtcacacag caaaaacaat 1980ggacaggccc agaggctgaa gcaaacagtg tcccttctgg ctgtgttgga gcttccccag 2040taaccaccta tttattttac ctctttccca aacctggagc atttatgcct aggcttgtca 2100agaatctgtt cagtccctct ccttctcaat aaaagcatct tcaagcttga aaaaaaaaa 2159113872DNAHomo sapiensmisc_feature(2663)..(2664)n=a, c, g or t 11gaaaccgaca caaatacctg aaatacacag ccacagacag acacacacgg aagcactcta 60tgcacaaaac actcacacag tacacaccat gctgcacata ccctgaccca aacagtctaa 120caagccctga gggtctccag ggctgccctg gggctattgc ccacccctcc caccgtcccc 180gctagggtga gatggtgttc cccagggaac agaagtctcc agtcccatct taagctctgc 240cggatcccgc gtgacatcag ctagccccct cgcggctgcc gggagctgtg agctctgtgc 300tggggccagg ccggcaccag gcacagacac ttaggccctt gttgggagaa cagagagagg 360ctctcttgtc cactgcctgt cttcggttcc aactgctggt tctcctagag gcctctcctc 420agactcgcag gtatgtggga ccagggaggc cgggtcctgg ccaaagggcc actggggtca 480gcccaggaga gggtgtggca gtgttgtggg ccgtttgcag gagcacacac gtctggcatt 540ggctaggggc aggctgcgct tccttagcag ttctgcagct tgctcttaag gcttggcagg 600gctgggcctc tcagggaagc ctgggctggg ggatcctctc agttcccctt cactttctct 660gttcccaaga aggccatgag gttggtgcct ccaggacccc cccttgtaaa gataggaaat 720ctctactcag agaggctggg ctgcagccca ggccccacag tgggccaaga ctaaggtctt 780gagatgcgcg gcaactgggc tttcaggtga gatctctgct cttcagcctt ttccaagcaa 840ggatgagact ttggggcccc aagcaatctg tttgcagggc ctgggcaccc tggccccttc 900tcccctgcag ggtggaagca aggaagacac tattcctggc cacatagatc agctggtcac 960accttctgtt gtttggcccc gaatagatat tggccagtct tgggtctctc tgtggcccca 1020gcccaaggct tccagggcag ctgcctttcc tgaggcattg ggcagaattc cttgtggcaa 1080ggagatcgta gcacagagcc cagctgggac tgcgcacagt aattcagggt tgccattgtt 1140cctctatggg agtccggaga gcccagcctg tgcttcacaa ggctatgtgg ccctaagaag 1200gtcctttttt aggccacagg ccttccatct gtgaaatggg ggatgggttc agactttatg 1260ccctgaaaag atccttccag ccctggccat cttggacttc tggagctacc ctggctcaca 1320ggggtcttgt tgccctgggt gtccccagtt cttgaaaaga atcagcctgg gaggggccac 1380accctgacca tcccccttta tcccttctga gatgtttgtt aggaagtctg ggtccagggg 1440atatcatttc ttgttccatc catgcagggg ttgcttacct cgggtaggaa accctcaggc 1500ggtggcaggt gcacaggtag gggaggatgg agagggcagt ggtgcctgaa gccctggatg 1560ggcggagctg accccccaac accaactcta tcatgcctgc tcctccctgt ccccccagag 1620ctgcctgatc attgctacag aatgaactct agcccagctg gtgaccccaa tgtccacagc 1680ccgtccaggg gccaaatggg aacatcaacc tggtgtgcct tcagccaacc caaatgccca 1740gcccacggac ttcgacttcc tcaaagtcat cggcagaagg gaactacgtg gaagtgtcct 1800actgtgccaa gcgcaagtct gatggggcgt tctatgcagt gaatggtact acagaaagaa 1860gtccatctta aatgaagaaa gagcagatgc cacatcatgg cagagcgcag tgtgcttctg 1920aagaacgtgc ggcacccctt cctcgtgggc ctgcgctact ccttccagac acctgagaag 1980ctctacttct gtgctcgact atgtcaacgg gggaggagct cttcttccac ctgcagcggt 2040gagcgccggt tcctggagcc cctgggccat gttctacgct gctgaggtgg ccagccgcca 2100ttggctacct gcactccctc aacatcattt acagggatct gaaaacagga gaaacattct 2160cttggactgc cagcccatgc cctccgtcat tctcagggac acgtggtgct gacggatttt 2220ggcctctgca aggaaggtgt agagcctgaa gacaccacat ccacattctg tggtacccct 2280gagtattgtg ccccctgaag tgcttctgga aagagcctta tgatcgagca gtggactggt 2340ggtgcttggg ggcagtcctc tacgagatgc tccatggcct gccgcccttc tacagccaag 2400atgtatccca gatgtatgag aacattctgc accagccgct acagatcccc ggatgccgga 2460cagtggccgc ctgtgacctc ctgcaaagcc ttctccacaa ggaccagagg cagcggctgg 2520gctccaaagc agactttctt tgagattaag aaaccatgta ttcttcagcc ccataaactg 2580ggatgacctg taccacaaga ggctaactcc acccttcaac ccaaatgtga caggacctgg 2640ctgacttgga agcatttttt ganncccaga gttcacccag gaagctgtgt ccaagtccat 2700tggctgtacc ccctgacact gtggccagca gctctggggc ctcaagctgc atttcctggg 2760attttcttat gcgccagagg atgatgacat cttggattgc tagaagagaa ggacctgtga 2820aactactgag gccagctggt attagtaagg aattaccttc agctgctagg aagagcgact 2880caaactaaca atggcttcat ccgagttagt caggtttatt gttattgcca gcatcatata 2940aagatgagaa tatatgtctc tacggaggtg ccatggatct ggcaggatca ggctcatcag 3000actacctcca cgaggactgt atctctgccc tgccaacctt gacaaatggc ttccaaatgt 3060ttaggtttgc ttacaaagat ggttactggg agctctaagc ctgccttatt ttggtgtttt 3120tagggaaggg aaaatgggag gaaaggggag aagagcaaag ggcgcttttt aaagagcttt 3180ccctaaaagc tccatccaat gagctttctg cttccatctc acttaaccac ccacccctac 3240ctgggaatgg aggcctggga gatgtggctt atttgctggg tacgtgacta tccctaataa 3300caaaggggtt ctgacactaa gacattaggg gagaatgttg ggtaggcagc cagcactctt 3360ttaccagagg gcctcctggt gtttggattt tgatctcaat gtgtaaacat gacagagatg 3420taacaagctc atagggtatc aatatctctt attgttctat gttgatgata tttgtctttg 3480ttgtgggtaa tactggacat tttgtttatt gggtctgggt gccttggtta tctgaacccc 3540cttcttgtct ccagagaacc ccctatttta tgagacttca tgggggggca ataactacct 3600ccacttaaga gtacctgaaa atgctagaca ctgactttcc cagcctcccc ttagctaggg 3660ccaggcatgg ggaccaggca taaacctgtg ccacattttg actcagggaa gggatcggga 3720gagctctttt gtgtggtaac tgtgataaca gtacccgcaa aattgagttc ctggtgtaga 3780agtgacaagg atgcaaactg tagcagttgg tgctcagtgg cagcaacgcc atcagaccag 3840ccctgcaatg tcattcctgg aagcctcaag tg 3872124728DNAHomo sapiens 12atggccagcc agcgggtaag cttccagcac gaggtgtacc cagcggagcc agccacaggc 60cctgcggccc ccagccagga gctggaggag cgaccgctgt cccgtcaggt gttcatcgtg 120caggagctgg aggtccgaga ccggctcgcc tcctcccaga tcaacaagtt cctgtaccta 180cacacgagtg agcggatgcc gcgacgtgcc cactctaaca tgctcaccat caaagcgctg 240catgtggccc ccactaccaa cctgggtggg cctgagtgct gtctccgcgt ctcgctgatg 300cccctgcggc tcaatgtgga ccaggatgcc ctcttcttcc tcaaggactt cttcactagt 360ctggtggccg gcatcaaccc cgtggtccca ggggagacct ccgctgaggc tcgccccgag 420actcgagccc agcccagcag ccccctggaa gggcaggccg aaggcgtaga gaccactggt 480tcgcaggagg ccccaggagg tggacacagc ccctcccctc ctgaccagca gcccatctac 540ttcagagagt tccgcttcac gtctgaggtc cccatctggc tggattacca tggcaagcac 600gtcacgatgg accaggtggg cacttttgct ggcctcctca tcggcctggc ccaactcaac 660tgctccgagc tgaagctaaa gcggctctgt tgcaggcacg ggctcctggg tgtggacaag 720gtgctgggct atgccctcaa cgagtggctg caggacatcc gcaagaacca gctgcccggc 780ctgctgggag gcgtgggccc catgcactcg gttgtccagc tcttccaagg gttccgggac 840ctgctgtggc tgcccattga gcagtacagg aaggatggcc gcctcatgcg ggggctgcag 900cgaggggctg cctcctttgg ctcatccaca gcctctgccg ccctggaact cagcaaccgg 960ttggtacagg ctatccaggc cacagctgag accgtgtatg acatcctgtc cccggcagcc 1020cccgtctccc gctccctgca ggataagcgc tctgcgcgga ggctgcgcag gggccagcag 1080cctgccgacc tgcgggaggg tgtggccaag gcctacgaca cagtgcgaga gggcatcttg 1140gatacagctc agaccatctg tgacgtggca tcgcggggcc atgagcagaa ggggctgacg 1200ggcgccgtgg ggggcgtgat ccgccagctg cccccgactg tggtgaagcc gctcatcctg 1260gccacggagg ccacgtccag cctgctcggg ggcatgcgca accagattgt ccccgacgcc 1320cacaaggacc acgccctcaa gactggcacc tgtcaccgga acctgtctgg gagggacgag 1380aacacgcttt gcaagaggaa gctctgcctc acagagccct gggctcactc agggaccctg 1440gccagcagct gcttcctctc cccacagcgg agagagaccc aagggtccca gggcggatgc 1500ttcccaccag gccagcccag cgtgcagggt ggcctccccc ccacacttct tcttagtctc 1560atcttcagct tcccatacga ggccatcctc atgaaatcag gcactgggag gtccctgggg 1620actgacaagt gccagctgtc ccttgctgtc tctctgcccc atggctgcag cagggaggga 1680aggagtgctg gcagcacacg gggcgccagg tgtgggcccc ggatgataag aagcctcggt 1740gaaaagacca tggacctggg gccacgaaga ctggggagcc cagcaactcc atgtggaagt 1800gcccactggt tccagtgggg ctgctgttat ctggggcgag ggccagtacc cacgaagaag 1860gagaggcagg taagcttcca gcacgaggtg tacccagcgg agccagccac aggccctgcg 1920gcccccagcc aggagctgga ggagcgaccg ctgtcccgtc aggtgttcat cgtgcaggag 1980ctggaggtcc gagaccggct cgcctcctcc cagatcaaca agttcctgta cctacacacg 2040agtgagcgga tgccgcgacg tgcccactct aacatgctca ccatcaaagc gctgcatgtg 2100gcccccacta ccaacctggg tgggcctgag tgctgtctcc gcgtctcgct gatgcccctg 2160cggctcaatg tggaccagga tgccctcttc ttcctcaagg acttcttcac tagtctggtg 2220gccggcatca accccgtggt cccaggggag acctccgctg aggctcgccc cgagactcga 2280gcccagccca gcagccccct ggaagggcag gccgaaggcg tagagaccac tggttcgcag 2340gaggccccag gaggtggaca cagcccctcc cctcctgacc agcagcccat ctacttcaga 2400gagttccgct tcacgtctga ggtccccatc tggctggatt accatggcaa gcacgtcacg 2460atggaccagg tgggcacttt tgctggcctc ctcatcggcc tggcccaact caactgctcc 2520gagctgaagc taaagcggct ctgttgcagg cacgggctcc tgggtgtgga caaggtgctg 2580ggctatgccc tcaacgagtg gctgcaggac atccgcaaga accagctgcc cggcctgctg 2640ggaggcgtgg gccccatgca ctcggttgtc cagctcttcc aagggttccg ggacctgctg 2700tggctgccca ttgagcagta caggaaggat ggccgcctca tgcgggggct gcagcgaggg 2760gctgcctcct ttggctcatc cacagcctct gccgccctgg aactcagcaa ccggttggta 2820caggctatcc aggccacagc tgagaccgtg tatgacatcc tgtccccggc agcccccgtc 2880tcccgctccc tgcaggataa gcgctctgcg cggaggctgc gcaggggcca gcagcctgcc 2940gacctgcggg agggtgtggc caaggcctac gacacagtgc gagagggcat cttggataca 3000gctcagacca tctgtgacgt ggcatcgcgg ggccatgagc agaaggggct gacgggcgcc 3060gtggggggcg tgatccgcca gctgcccccg actgtggtga agccgctcat cctggccacg 3120gaggccacgt ccagcctgct cgggggcatg cgcaaccaga ttgtccccga cgcccacaag 3180gaccacgccc tcaagactgg cacctgtcac cggaacctgt ctgggaggga cgagaacacg 3240ctttgcaaga ggaagctctg cctcacagag ccctgggctc actcagggac cctggccagc 3300agctgcttcc tctccccaca gcggagagag acccaagggt cccagggcgg atgcttccca 3360ccaggccagc ccagcgtgca gggtggcctc ccccccacac ttcttcttag tctcatcttc 3420agcttcccat acgaggccat cctcatgaaa tcaggcactg ggaggtccct ggggactgac 3480aagtgccagc tgtcccttgc tgtctctctg ccccatggct gcagcaggga gggaaggagt 3540gctggcagca cacggggcgc caggtgtggg ccccggatga taagaagcct cggtgaaaag 3600accatggacc tggggccacg aagactgggg agcccagcaa ctccatgtgg aagtgcccac 3660tggttccagt ggggctgctg ttatctgggg cgagggccag tacccacgaa gaaggagagg 3720caggtgctgg ccagcagacc agccaggact accgtggcga cgctcccagg ccagatggtg 3780gcgggtagtg gagggctgtc tggtgggctg ccgagaccga gtgcacaggg ctctgaccta 3840tgaattgaca gccagtgctc tcgtctcccc tctggctgcc aattccatag gtcacaggta 3900tgttcgcctc aatgccagcc accaggacct gcagggatag gggagggccg ggggtgtcca 3960gcagtcagca gagatcctgc gaccccagtg cagcactcat ggtcccacct ccctctgtct 4020cattccccgt gaatgagcct gaacagcttc agtcctgccc ctgccctgcc tgccctgtgg 4080cacctctatg ctttgcccat gctgttccct tgggctgcaa tactcttcct agcttatttg 4140ccaggctcac tcttactaac cctttcaagc tctgtccaag catttgctgc ctccagaagg 4200ccttattgaa gcttctaagt ccccacctgg gcacccccac acagtgctgc cgcagagcac 4260tgccctctcg gagccccggg tgctggtttc tgcttatgtc tcgactcctc ttccccatct 4320gtgagctcag ttcccagccc aaggcgcgtg cccaaataaa tgtttgctga accaatcctg 4380agcctctgtc ttgcaacctg aggaagcaac ccaccgaaca atgcagtgtg gccaaagggg 4440ggctgagtgc tctaggccca gtgtttgtgc ttggagcccc cccacccagg atggggccct 4500gagccagcct ccccatctgc ttcctactct cccctccttt gccagtctca tctccctgga 4560gcacagccct gtggttggtg gagcagcttc tccagcccct aggattccta agagggccca 4620ggaccccagc tgctggtaga ggaagagcag ccaacccagg acaggacagc tgaccccacc 4680cctgtcccgc ctcccacaac agcctcattt ccacctattt ctttgtgg 4728136650DNAHomo sapiensmisc_feature(4298)..(4298)n=a, c, g or t 13tcctccacat accggctcag ctcctccagg acgcagcccg ccagacacgc tgtggaagct 60gaggacccgg ccttgttttg ttcatgaaca ttgggtttag tgcctggcaa cttgatgcat 120atggaagagc aatgccaagt gatctgacat aatacaaatt cacgaagtga cattcaatca 180caagcaaagt tggaaattcc aaagagaagt ggtgagatct ttactagtca cagtgaagat 240gggagaaaat gacatacctg cagcagatgt gggctgaaaa tatcctcttc tctgcccaat 300caggaatgct acctgttttt gggaataaac tttagagaaa ggaagggcca aaactacgac 360ttggctttct gaaacggaag cataaatgtt cttttcctcc atttgtctgg atctgagaac 420ctgcatttgg tattagctag tggaagcagt atgtatggtt gaagtgcatt gctgcagctg 480gtagcatgag tggtggccac cagctgcagc tggctgccct ctggccctgg ctgctgatgg 540ctaccctgca ggcaggcttt ggacgcacag gactggtact ggcagcagcg gtggagtctg 600aaagatcagc agaacagaaa gctattatca gagtgatccc cttgaaaatg gaccccacag 660gaaaactgaa tctcactttg
gaaggtgtgt ttgctggtgt tgctgaaata actccagcag 720aaggaaaatt aatgcagtcc cacccgctgt acctgtgcaa tgccagtgat gacgacaatc 780tggagcctgg attcatcagc atcgtcaagc tggagagtcc tcgacgggcc ccccgcccct 840gcctgtcact ggctagcaag gctcggatgg cgggtgagcg aggagccagt gctgtcctct 900ttgacatcac tgaggatcga gctgctgctg agcagctgca gcagccgctg gggctgacct 960ggccagtggt gttgatctgg ggtaatgacg ctgagaagct gatggagttt tgtgtacaat 1020gaaccgaaaa ggcccatgtt gaggattgac gctgagagga gcccccggtc gtggccagca 1080ttatgcatgt gtggatccta actgacatgt ggtgggcacc atctttgtga tcatcctggc 1140ttcggtgctg cgcatccggt gccgcccccg ccacagcagg ccggatccgc ttcagcagag 1200aacagcctgg gccatcagcc agctggccac caggaggtac caggccagct gcaggcaggc 1260ccggggtgag tggccagact cagggagcag ctgcagctca gcccctgtgt gtgccatctg 1320tctggaggag ttctctgagg ggcaggagct acgggtcatt tcctgcctcc atgagttcca 1380tcgtaactgt gtggacccct ggttacatca gcatcggact tgccccctct gcgtgttcaa 1440catcacagag ggagattcat tttcccagtc cctgggaccc tctcgatctt accaagaacc 1500aggtcgaaga ctccacctca ttcgccagca tcccggccat gcccactacc acctccctgc 1560tgcctacctg ttgggccctt cccggagtgc agtggctcgg cccccacgac ctggtccctt 1620cctgccatcc caggagccag gcatgggccc tcggcatcac cgcttcccca gagctgcaca 1680tccccgggct ccaggagagc agcagcgcct ggcaggagcc cagcacccct atgcacaagg 1740ctggggaatg agccacctcc aatccacctc acagcaccct gctgcttgcc cagtgcccct 1800acgccgggcc aggccccctg acagcagtgg atctggagaa agctattgca cagaacgcag 1860tgggtacctg gcagatgggc cagccagtga ctccagctca gggccctgtc atggctcttc 1920cagtgactct gtggtcaact gcacggacat cagcctacag ggggtccatg gcagcagttc 1980tactttctgc agctccctaa gcagtgactt tgacccccta gtgtactgca gccctaaagg 2040ggatccccag cgagtggaca tgcagcctag tgtgacctct cggcctcgtt ccttggactc 2100ggtggtgccc acaggggaaa cccaggtttc cagccatgtc cactaccacc gccaccggca 2160ccaccactac aaaaagcggt tccagtggca tggcaggaag cctggcccag aaaccggagt 2220cccccagtcc aggcctccta ttcctcggac acagccccag ccagagccac cttctcctga 2280tcagcaagtc accggatcca actcagcagc cccttcgggg cggctctcta acccacagtg 2340ccccagggcc ctccctgagc cagcccctgg cccagttgac gcctccagca tctgccccag 2400taccagcagt ctgttcaagt tgcacagaat ccacgcctct tctgccgcga cacctcacac 2460gaggaaaagg acggggcggg tccctcctga gcccacccct gggccctcgg ccaccacgga 2520tgcaacatgt gcacccagta cttgccagat ttttccccat tacaccccca gtgtgcgcag 2580atccttggtc cccagaggca caccccttga actgtggacc tccaggcctg gaacacgagg 2640ctgctaccag aaaaccccag gcccctgtta ctcaaattca acagccagtg tggtcgtgcc 2700tgactcctcg accagcccct ggaaccacat ccacctgggg aggggccttc tgcaatggag 2760ttctgacacc gcagagggca ggccatgccc ttatccgcac tgccaggtgc tgtcggccca 2820gcctggctca gaggaggaac tcgaggagct gtgtgaacag gactgtgtga gatgttcagg 2880cctagctcca accaagagtg tgctccagga tgtttttggg cccctacctg gcacagagtc 2940ctgctccgtg gtgaaatgga atggaccaca gcaaacacca ttcttttggc cgtacttcct 3000aggaagcact gggaagagga ctggatgatg gtgggagggt gagagggtgc cgtttcctgc 3060tccagctcca gaccttgctc tgacgcaaaa catctgcaga tgccagcaac atccatgtcc 3120agccaggaca accagctgct gcctgtggcg tgtgtgggct ggatcccttg aaggctgagt 3180ttttgaaggg cagaaagcta gctatgggta gccaggtgtt tccaaaggtg ctgctccttc 3240tccaacccct acttggtttc cctacacccc aatgcctcat gttcatacca gccaagtggg 3300ttcagcagaa acgcatgaca cctttatcac ctcccttcct tgggtagagc tcgtgagaca 3360ccagcgtttg gccccctcca cagtaaggct gctacatcag gggcaaccct ggctctatca 3420ttttcctttt ttgcctaaag gaccagtagg cataggtgag ccctgagcac taaaaggagg 3480gggtccctgg aagctttccc agctatagtg tgggagttct gttccctgga gggtggggta 3540cagcagcctt tggttcctct gggggttgag aataagaaat agtggggtag ggaaaaactc 3600ctctttgaag atttcctgtc tcagagtccc tgagtagtta gaaaggagga atttctgctg 3660ggcctttatt ctggggcaag aggaaaggat gggaattaag ggtagaaaga ggcaaaaatt 3720tccagttgag cgggggccaa caaaaagttt ttttttttgg aaaaagtttt tttcttagaa 3780caaggatggc aaaatgggtg caccagcaat aggaaagagt caaacgtgtg aacccttggg 3840gtttgggaca ggcccatgag gccccagctc ccctagtata agccatacag gtccaaggga 3900tcctcacagt gagagtggac ttagagcacg aagtcgtggc gctgcgatct gagtgcgacc 3960aagagtctga tagggcctag atgcagggta gacaatctca gcgccacagg gcagtcctga 4020cccactcttt ggcccctcag cgcacttatc ccactttgga aatgtgaatt gtggtgggca 4080aaagttgggg caagaggacc cccaactggg aaactttttc ccctccaggt tagttgggga 4140actagcaccc tcaggtaacc caccactggc gtaatttata tctgaaccca gaccagacgc 4200tttgaatcag gcactaaact ccagaaatat atttatttgc taatatattt atccacaaat 4260gtggtctggt cttgtggttt tgttctgtcg tggagctngt ccagctngca ngngngtaga 4320gcaagcngtc catgcgttcg ttgtcgtaca tctaagagaa gtaaattatt tatgttatca 4380gaggctaggc tccgattcat gaaatggata gggtagagta gaggggcttg gccaattaag 4440aactggtttg taagccccta aaagtgtggc ttaagtgaag atcagggaaa ggaagaaagc 4500catgaactgg aatccttaac tgtgccttca gtctattatt attatactgt tcacttcaca 4560cattatccat acttcaggtg gactcagacc tggggcaaat actctgtggc ctcgcttttt 4620cagtccataa aatgggccta cttaatagtt gttagcagga ctatacatga gataatagag 4680tgtagaaaga tatgttccaa aagtggaaaa gttttattca agtgatagaa gaacatccaa 4740acctgtcaca agaagcccat ctgaaacaca gcatgggacc gccaacaaga agaaagcccg 4800cccggaagca gctcaatcaa ggaggctggg ctggaatgac agcgcagcgg ggcctgaaac 4860tatttatatc ccaaagctcc tctcagataa acacaaatga ctgcgttctg cctgcactcg 4920ggctattgcg aggacagaga gctggtgctc cattggcgtg aagtctccag gggccagaaa 4980ggggcctttg tcgcttcctc acaaggcaca agttcccctt ctgcttcccc gagaaaggtt 5040tgggtagggg gtgggtggtt tagtgcctat agaacaaggc atttcgcttc ctagacggtg 5100aaatgaaagg gaaaaaaagg acacctaatc tcctacaaat ggtctttagt aaaggaaccg 5160tgtctaagcg ctaagaactg cgcaaagtat aaattatcag ccggaacgag caaacagacg 5220gagttttaaa agataaatac gcattttttt ccgccgtagc tcccaggcca gcattcctgt 5280gggaagcaag tggaaaccct atagcgctct cgcagttagg aaggaggggt ggggctgtcc 5340ctggatttct tctcggtctc tgcagagaca ataccagagg gagagcagtg gattcactgc 5400ccccaatgct tctaaaacgg ggagacaaaa caaaaaaaaa caaacgttcg ggttaccatc 5460ggggaacagg accgacgccc agggccacca gcccagatca aacagcccgc gtctcggcgc 5520tgcggctcag cccgacacac tcccgcgcaa gcgcagccgc ccccccgccc cgggggcccg 5580ctgactaccc cacacagcct ccgccgcgcc ctcggcgggc tcaggtggct gcgacgcgct 5640ccggcccagg tggcggccgg ccgcccagcc tccccgcctg ctggcgggag aaaccatctc 5700ctctggcggg ggtaggggcg gagctggcgt ccgcccacac cggaagagga agtctaagcg 5760ccggaagtgg tgggcattct gggtaacgag ctatttactt cctgcgggtg cacaggctgt 5820ggtcgtctat ctccctgttg ttcttcccat cggcgaagat ggccctggag acggtgccga 5880aggacctgcg gcatctgcgg gcctgtttgc tgtgttcgct ggtcaaggtg tcagtcgggg 5940acctggttgt agggcccatg ggggaccaag gtcggggaaa gagggcggaa tggggctcgt 6000aggatcgcgg acaggtcttg cagctgaggg caggggcggt cttacatgcc tttgaatcct 6060cagctcttag acgttcggtg aacttacgtt ggagccgaaa gacactggga gtcagaggcg 6120ggtggggatc cgctgctgag tgagtagtcg gaaaggatgc ctgaccctga gtagactcac 6180agaactgttt cttttcctgc ttcaggaatc gtgcgggagc tgaaaagtcg aggagtggcc 6240tcactgggtc agcatgacga tcaagcgaga ttcagattga gtgtgtttca tcaagttctc 6300tagctgcctg ggctgcctcc cttccctcgg ccccgagtgc agaacgtgga ggtgaacggg 6360atgaatccaa gctggttcgc agggcagtcc tcactgagca gtctctttcc aactctcacc 6420accttttcca gctggtcctg ggatgtgagg aatcctgttg ggggcaggag gctggcagga 6480ggaaatagat agctctttgc cccttgtttc cagacaagat aaggggagaa ttctactaga 6540gccattccta gccaccctgc cttctctgca ttttgggagg tgtgccctcg agccagctga 6600gaagatacca tggctgcctg ggggctgggc aggatttgga acacctcgtg 6650141206DNAHomo sapiens 14gcagtgccag gacctctccc ggaggcgggg cagagcagca gcttctcggc cctgtgccga 60gcccaggcct gcacccctaa ggcaggcact gctccgtgat ccaggaacca cctctctcta 120cagctgggag tgagcagtca gagagggaga cagccttgcc cggtgctacc cagcaagcta 180gtcaccgagt gggcagaggg aggagcggcc ctcaccggat gtcaagcagc ctgggtcccc 240agtccagctc tgcctgtccc tcgcaataac gcctcagtga cgaccatttg tgagccatct 300ctctgtctca ggcacggtgc tacatgccaa cgaaacctgc tcccattgaa ccctggccag 360ccagtgaaga aagggttggg cctgggaggt gccactttac agacaggggc accaaggggc 420agggtggcag gaggcccacc ggacgttccc catgaagtag cagtcccagc atccacaccc 480agcaggcacc acgctggccc gcagcctccc tgccagcacg cctggcttcc cggcctcgga 540acttgatctg ctccctcttc cggacactgg ggctcctgcc aagtcctggg ctgggcagca 600actgctgaac attctaagaa atccctccca gggttttctc aggagcccgg gtggggcagg 660aagtccccag gggctgaggg gaccgtggcg gcaggtggca cccagagcag cactctcctg 720gggcccaggc tgttgggcca gaggcaggac tgtgaggcct agtgtagggc ctcctgccag 780tggccggcac ctacttgtgg ggctgggggt tcccccagca ggttgggctc cccacctgac 840acactcacag accttgtgcc ttggagagcc agtgttcccg gggccacata gctatgccgc 900ccaggggctg ggcctgtccc agctctggtc ccccggcccc aggtcctgga cgctggtccg 960cgcagcagca ggcggcctcc ggaggacacg atgtgactgg ctgccgctac gtcgcactca 1020gatgagtctg cgccggatcg acctgctgcc gagtcctgcc ggacaggcac aggcagggag 1080tgaaaattat ctaccccttt ttatttctta ataactgaat gaaaataaac attggtggtt 1140tgacaaataa ctacatattt tcaaacccag ccagtccagg ggatgcagtt tccaggtgcg 1200ttatgc 1206151443DNAHomo sapiens 15gccttttatc actgacccaa agcgaaaagc accaggttta actctgttcc ccctgtgcta 60ggtccccaca ggttttgtta tcctgtatcc ttccttactc ctagcagcta ctctgatcga 120ttttctctca ccctcagagc agacttgtgg ccttgtttgg ggaagcactg gaattttgaa 180cccccagcct atttgggtca attgtttggc aagagtgtcc gcttcatgat gctggtgatg 240gcatgcacct cgtcacatgt gcacggctag gcttgtgcag gtggcctcta ttacccaaac 300actgaaggga agcccctctg tgtccttgga gagatgccag gtgcttagtt tacatttttg 360cctgcttgga gagctaacag cttgaagtaa accaatccat cagggactcc tgaggttttc 420accagccagc accacccaat cgtgcgtgaa gactttctga ctccctggac attgccatgg 480actcaacctg tcacttcagg acctgttttt gaactaacaa agctagactt ctgattctct 540cttgcctgca cctacctgta cattccgaac acatggtaga gactctacaa aatgcttaat 600atgtgatcta tggacggttc cccctgaaat tataaatgct gccatcttca tccttctggt 660tttcccaagc tattacccct atccatttgt ctgtggtata caacgtcact atccaggcct 720ccgtctcgga actgtgtgaa gctctttggt ctagggacca aaggcaggaa ttatttagtg 780atcagacaat aagaaaacac tgaaagagat gatttgcctt tgatggatgt aaaaatacta 840aaaatttatt ttcaatttat ggtaatgcta cttagccatt ttctctcaaa caccactgga 900gaatttatat aacatgaagc atatacaaaa tgcatctagg gggtaatgag gcttctcttt 960catcaacttc tgccttttag gatttgcccc aatattgtac ttggaggtaa atattaaaac 1020tccattgagg actggtataa agttgtaaag tgaacaaaac ccagtagaaa gctattgata 1080aagaatctat tttataaaat aagttttata caataaaatc tactctgtaa ttaccttttc 1140aaagtatatt tctaaaatag cttatatgcc cttctgtacc aaattttcta aataagggat 1200tatgttcaca ctttctcagt cctccttcca gctcttcaac ctactatccc aataagggtc 1260ataagactga ggcagtttca acagctcctg ctaaggttaa agaaagatac ggggaagcat 1320catgaaagga taggactctc cctatctaat gtatgtttat acatacctta tatatggagg 1380ctaataagtt tcctttaagt atatcaataa ttaagatctg tactaagtga ccactataag 1440tgt 1443161957DNAHomo sapiens 16gcggccgccg agctccgcgc ggggcaaacc tcccggcgcg gccatgcggg gaggtaagtg 60atctgcctgt gcgcccaggg cgtgggaagg cgcccgccct ctcctctctc caggatgaaa 120ggaaacgaag aatgccgcaa tgaaaaccgc tctgccctcc caaaaacaca tcttggccgt 180gtgtccggtg ctcctgcagc tcgttgcacc cacggacgtg ggctctcact gtggagtgga 240gtgggggcag aagcgtgccc tgccccacgg agagccccgg ctcgcctggg gctgctggca 300gtgctcgggg agcgggacgg ggtggtggca cgactcggcg gtgaccccga gaacgccaca 360cctccaccct ccactttcca aagaccggct tccccgggga gcccccacac taaacgccag 420cgaactgcct ctccgtgaaa gtcttagcca gaaactttcc ccgctttgtc gccagtgcca 480cagagagtcg tgtggctctg ggccggcgct gctggtccaa gaggcagcct ggcgtcttct 540gcccctaccg tccccttctc aggccagttc tcacttgccc ctgagacgcc attcccggct 600cggtgaaaaa ggcactatat ccatccctgc atcgtctcca agactcattc cctctaaacc 660ttcaagttcc atggaaaatg ggagaccacc tgatcctgca gactgggccg tgatggatgt 720cgtcaattat ttccgaaccg tgggatttga ggagcaagct agtgcttttc aggaacagga 780aattgatgga aaatccctgc tattgatgac aagaaatgat gtgttgacag gacttcagtt 840aaaattgggg cctgctctga aaatctacga atatcatgta aaacctctgc agacaaagca 900tttaaagaac aactcttcat agtacagtca aattggggtc ttcgacctca aaaaaaatac 960ataatgacat aattcagttt catgtaatga aactttgtaa acagaataca tacatgtgta 1020tatgtaaaga atttcaatca aatgaaacgt tatcctattg gatagactag gcaattcatc 1080agctcacctg aaatcagcca ggaggagcaa ggacaagatg cgcacagggt ggttttcctc 1140atggattttg tcaaatagat gatctttgac acgattagac actcctcccc acaaaggctt 1200tgaaatcata aggattttcc tcatctcttt atagctttcc caaaatcttt taaaaaaaga 1260atttaattaa atgacagtct tttggttaca gacttaggat gagtaaaaac aagaaaattt 1320ggggaggggg agaaagaaga aagggattgc tgtctccctt gaattcctct gttccttaga 1380gcttgtgtta cttggacgga attgccaaca ccctttttta tagagggttc tccacttgac 1440cttattaagg ttttattggg atatgctgca gtgtttgaaa tgaacatgca tcatggcccc 1500ttcaggagca gaatcatagc tctgaaaaga gaagctccgt tgtgtactga ggatatccat 1560ccatattcag ctagctttca aatggggtgt aatgatattt tctgcataga ttttctttta 1620aattggttct ttgtttctga agaaagaatt ttttttaact tcatggtttt atttataata 1680atttgtttct gaagaaattt gccgagagtt acaggtcaaa aagccttgtt actagtacag 1740aatattttta tatatattcc ttcatgatgg tgtaattttt tttaattgtc ctatgctttg 1800ttcggttcct gggttaagta cttgttttta agagcttgga aaaagtgggc ttgctacatc 1860tctgttcaaa gagacatttg ttcaatctct gtgtgtcaac gccttgttga attggtgctt 1920tgtggtagca ataaagcatt gcttcagttt ataaaaa 1957172074DNAHomo sapiens 17tgcagctatt ttaggttctc taacttcatc gtagtttata gggtaagtaa agggaagggg 60aaagtgattg gtgtggttgt ctcccataag aactgatttt tttctactga agcatgtata 120aagtttatat atgacttttt atatttgttt aataaaaatt ttacaggaac taaatttgat 180tatcaatatg aagtttttct ttaatttcag atttcaacta ttgcagaaag tgaagattca 240caggagtcag tggatagtgt aactgattcc caaaagcgaa gggaaattct ttcaaggagg 300ccttcctaca gggagaagtc tgaagaggag acttcagcac ctgccatcac cactgtaacg 360gtgccaactc caatttacca aactagcagt ggacagtata ttgccattac ccagggagga 420gcaatacagc tggctaacaa tggtaccgat ggggtacagg gcctgcaaac attaaccatg 480accaatgcag cagccactca gccgggtact accattctac agtatgcaca gaccactgat 540ggacagcaga tcttagtgcc cagcaaccaa gttgttgttc aaggtactca aaaattgtaa 600agcaggatgt cagtgaattt gaattctgaa cgtcagtttg aagatggtaa catgtttagt 660atataaatct tttccactca aaccatacat tttaattgat attaataatt aatatgaact 720aattttataa agaccttcaa atttttttaa gtaacattag gttccttatt aggagagcat 780attattacgc tgtttttaga agcagtttga caaatagtga ttgtgtttgt ttttacaaat 840ggtgaatcag ttagaaaaat aaaacttcag tttatttagc cattatcatt tacattaaaa 900caatatgttt ttcaaataat ataattggca tcaagtgata cactttttca tacttttagt 960tttgttttaa ttcaaaattt ataatagttg accataatgc tttatcttct ttttcatttt 1020gctcatttta tgaaaaatca tggtcgtttt ttatgtctgt ggcaagagtc tacttgatat 1080ttgtttaata tgaattttac caatatcaaa ggtatagtac tactgaggaa ctatactcta 1140tctaggtaag atcatccaat gtctgtgccc catctgtacc ttttagaccg taagcgtgcc 1200tctggagacg tacaatacta taccagtatt cgctactagc taccctacta gctactattg 1260gcccctggag ttgttatggc atcctcccct agctacttcc tacacagcct gtctgaagat 1320agcagctacg tataagtaga gaggtccgtc taatgaagat acagggaagc tagttctaga 1380gtgtcgtaga aagaagtaaa gaatatgtga aatgtttaga aaacagagtg gctagtgcgt 1440tgaaaatcaa taactagaca ttgattgagg agcttaaagc acttaaggac ctttactgcc 1500acaaatcaga ttaatttggg atttaaattt tcacctgtta aggtggaaaa tggactggct 1560tggccacaac ctgaaagaca aaataaacat tttattttct aaacatttct ttttttctat 1620gcgcaaaact gcctgaaagc aactacagaa tttcattcat ttgtgctttt gcattaaact 1680gtgaatgttc cagcacctgc ctccacttct cccctcaaga cattttcaac gccaggaatc 1740atgaagagac ttctgctttt caaccccacc ctcctcaaga agtaataatt tgtttacttg 1800taaattgatg ggagacatga ggaaaagaaa atctttttaa aaatgatttc aaggtttgtg 1860ctgagctcct tgattgcctt agggacagaa ttaccccagc ctcttgagct gaagtaatgt 1920gtgggccgca tgcataaagt aagtaaggtg caatgaagaa gtgttgattg ccaaattgac 1980atgttgtcac attctcattg tgaattatgt aaagttgtta agagacatac cctctaaaaa 2040agaactttag catggtattg aggacttaga aatg 207418933DNAHomo sapiens 18atggcggagg ctgtactgag ggtcgcccgg cggcagctga gccagcgcgg cgagtcttcg 60agctcccatc ctcctgcggc agatgttcga gcctgtgagc tgcaccttca cgtacctgct 120gggtgacaga gagtcccggg acgccgttct gatcgaccca gtcctggaaa cagcgcctcg 180ggatgtccag ctgatcaagg agctggggct gcggctgctc tatgctgtga atacccactg 240ccacgcggaa ccacattaca ggcttggggc tgctccgttc cctcctccct ggctgccagt 300ctgtcatctc ccgccttagt ggggcccagg ctgacttaca cattgaggat gggagactcc 360atccgcttcg ggcgcttcgg tacagcccca ctcctggctg ctttcacggg ctggtgtgga 420gtatctgtgg cttttccagg cacatggtgc aagctctcgg tggatctaac actctgggtt 480ctggagggcg atggccctct tctcacagct ccactagggg cagtgcccca gtgggaactc 540tctgcgttgg agaccagggc cagccctggc cacaccccag gctgtgtcac cttcgtcctg 600aatgaccaca gcatggcctt cactggagat gccctgttga tccgtgggtg tgggcggaca 660gacttccagc aaggctgtgc caagaccttg taccactcgg tccatgaaaa gatcttcaca 720cttccaggag actgtctgat ctaccctgct cacgattacc atgggttcac agtgtccacc 780gtggaggagg agaggactct gaaccctcgg ctcaccctca gctgtgagga gtttgtcaaa 840atcatgggca acctgaactt gcctaaacct cagcagatag actttgctgt tccagccaac 900atgcgctgtg gggtgcagac acccactgcc tga 93319525DNAHomo sapiens 19gccatgggtt ccccttcagc ctgtccatac agagtgtgca ttccctggca ggggctcctg 60ctcacagcct cgcttttaac cttctggaac ctgccaaaca gtgcccagac caatattgat 120ggtgtgccgt tcaatgtcgc agaagggaag gaggtccttc tagtagtcca taatgagtcc 180cagaatcttt atggctacaa ctggtacaaa gggcaaaggg tgcatgccaa ctatcgaatt 240ataggatatg taaaaaatat aagtcaagaa aatgccccag ggcccgcaca caacggtcga 300gagacaatat accccaatgg aaccctgctg atccagaacg tcacccacaa tgacgcagga 360atctataccc tacacgttat aaaagaaaat cttgtgaatg aagaagtaac cagacaattc 420tacgtattct atgagtcagt acaagcaagt tcacctgacc tctcagctgg gaccgctgtc 480agcatcatga ttggagtact ggctgggatg gctctgatat agcag 52520377DNAHomo sapiensmisc_feature(28)..(28)n=a, c, g or t 20ctcaaccaac atctgacatc tttcccgngg agcaacttcc tgctccacgg gaaagaggcc 60gaaggattta cccntggacc cataagtctg ancatcctgc tgaagtcccc tcnccattgc 120tccttnaagc caaanctaca ctttgctggt tcctgtcccc tctgagaaag gggatagaaa 180gctccttcct ctatgtcctc ccatcgagat ctgttctggg gatggagctt ccaacttcct 240cttgcagcag gaaagaatgc tgctcaccct tctgtcttgc agagtgggat tgtgggaggg 300attggcagcc ttcttctcca ccacctgtcc agcttcttcc tggtcagggc tgggaccccc 360aggaatatta tgttgcc 37721709DNAHomo sapiens 21tctgaatgtt ttggtgaata aatctgttct tcagcaaccc tacctgcttc tccaaactgc 60ctaaagagat ccagtactga tgacgctgtt cttccatctt tactccctgg aaactaacca
120cgttgtcttc gtttccttca ccacgcacca ggagctcaga gatcaaagcg gctttccatc 180ttgttctccc agccccagga cactgactct gtacaggatg gggccgtcct cttgccctcc 240ttctcatcct aatccccctt ctccagctga tcaacccggg gagtactcag tgttccttag 300actccgttat ggataagaag atcaaggatg ttctcaacag tctagagtac agtccctctc 360ctataagcaa gaagctctcg tgtgctagtg tcaaaagcca aggcagaccg tcctcactgc 420cctgctgggg atggctgtca ctggctgtgc ttgtggctat ggctgtggtt cgtgggatgt 480tcagctggaa accacctgcc actgccagtg cagtgtggtg gactggacca ctgcccgctg 540ctgccacctg acctgacagg gaggaaggct gagaactcag ttctgtgacc atgacagtaa 600tgaaaccagg gtcccaacca agaaatctaa ctcaaacgtc ccacttcatt tgttccattc 660ctgattcttg ggtaataaag acaaactttg tacctctcaa aaaaaaaaa 709223195DNAHomo sapiens 22gccaggaata actagagagg aacaatgggg ttattcagag gttttgtttt cctcttagtt 60ctgtgcctgc tgcaccagtc aaatacttcc ttcattaagc tgaataataa tggctttgaa 120gatattgtca ttgttataga tcctagtgtg ccagaagatg aaaaaataat tgaacaaata 180gaggatatgg tgactacagc ttctacgtac ctgtttgaag ccacagaaaa aagatttttt 240ttcaaaaatg tatctatatt aattcctgag aattggaagg aaaatcctca gtacaaaagg 300ccaaaacatg aaaaccataa acatgctgat gttatagttg caccacctac actcccaggt 360agagatgaac catacaccaa gcagttcaca gaatgtggag agaaaggcga atacattcac 420ttcacccctg accttctact tggaaaaaaa acaaaatgaa tatggaccac caggcaaact 480gtttgtccat gagtgggctc acctccggtg gggagtgttt gatgagtaca atgaagatca 540gcctttctac cgtgctaagt caaaaaaaat cgaagcaaca aggtgttccg caggtatctc 600tggtagaaat agagtttata agtgtcaagg aggcagctgt cttagtagag catgcagaat 660tgattctaca acaaaactgt atggaaaaga ttgtcaattc tttcctgata aagtacaaac 720agaaaaagca tccataatgt ttatgcaaag tattgattct gttgttgaat tttgtaacga 780aaaaacccat aatcaagaag ctccaagcct acaaaacata aagtgcaatt ttagaagtac 840atgggaggtg attagcaatt ctgaggattt taaaaacacc atacccatgg tgacaccacc 900tcctccacct gtcttctcat tgctgaagat cagtcaaaga attgtgtgct tagttcttga 960taagtctgga agcatggggg gtaaggaccg cctaaatcga atgaatcaag cagcaaaaca 1020tttcctgctg cagactgttg aaaatggatc ctgggtgggg atggttcact ttgatagtac 1080tgccactatt gtaaataagc taatccaaat aaaaagcagt gatgaaagaa acacactcat 1140ggcaggatta cctacatatc ctctgggagg aacttccatc tgctctggaa ttaaatatgc 1200atttcaggtg attggagagc tacattccca actcgatgga tccgaagtac tgctgctgac 1260tgatggggag gataacactg caagttcttg tattgatgaa gtgaaacaaa gtggggccat 1320tgttcatttt attgctttgg gaagagctgc tgatgaagca gtaatagaga tgagcaagat 1380aacaggagga agtcattttt atgtttcaga tgaagctcag aacaatggcc tcattgatgc 1440ttttggggct cttacatcag gaaatactga tctctcccag aagtcccttc agctcgaaag 1500taagggatta acactgaata gtaatgcctg gatgaacgac actgtcataa ttgatagtac 1560agtgggaaag gacacgttct ttctcatcac atggaacagt ctgcctccca gtatttctct 1620ctgggatccc agtggaacaa taatggaaaa tttcacagtg gatgcaactt ccaaaatggc 1680ctatctcagt attccaggaa ctgcaaaggt gggcacttgg gcatacaatc ttcaagccaa 1740agcgaaccca gaaacattaa ctattacagt aacttctcga gcagcaaatt cttctgtgcc 1800tccaatcaca gtgaatgcta aaatgaataa ggacgtaaac agtttcccca gcccaatgat 1860tgtttacgca gaaattctac aaggatatgt acctgttctt ggagccaatg tgactgcttt 1920cattgaatca cagaatggac atacagaagt tttggaactt ttggataatg gtgcaggcgc 1980tgattctttc aagaatgatg gagtctactc caggtatttt acagcatata cagaaaatgg 2040cagatatact taaaagttcg ggctcatgga ggagcaaaca ctgccaggct aaaattacgg 2100cctccactga atagagccgc gtacatacca ggctgggtag tgaacgggga aattgaagca 2160aacccgccaa gacctgaaat tgatgaggat actcagacca ccttggagga tttcagccga 2220acagcatccg gaggtgcatt tgtggtatca caagtcccaa gccttccctt gcctgaccaa 2280tacccaccaa gtcaaatcac agaccttgat gccacagttc atgaggataa gattattctt 2340acatggacag caccaggaga taattttgat gttggaaaag ttcaacgtta tatcataaga 2400ataagtgcaa gtattcttga tctaagagac agttttgatg atgctcttca agtaaatact 2460actgatctgt caccaaagga ggccaactcc aaggaaagct ttgcatttaa accagaaaat 2520atctcagaag aaaatgcaac ccacatattt attgccatta aaagtataga taaaagcaat 2580ttgacatcaa aagtatccaa cattgcacaa gtaactttgt ttatccctca agcaaatcct 2640gatgacattg atcctacacc tactcctact cctactccta ctcctgataa aagtcataat 2700tctggagtta atatttctac gctggtattg tctgtgattg ggtctgttgt aattgttaac 2760tttattttaa gtaccaccat ttgaacctta acgaagaaaa aatcttcaag tagacctaga 2820agagagtttt aaaaaaacaa aacaatgtaa gtaaaggata tttctgaatc ttaaaattca 2880tcccatgtgt gatcataaac tcataaaaat aattttaaga tgtcggaaaa ggatactttg 2940attaaataaa aacactcatg gatatgtaaa aactgtcaag attaaaattt aatagtttca 3000tttatttgtt attttatttg taagaaatag tgatgaacaa agatcctttt tcatactgat 3060acctggttgt atattatttg atgcaacagt tttctgaaat gatatttcaa attgcatcaa 3120gaaattaaaa tcatctatct gagtagtcaa aatacaagta aaggagagca aataaacaac 3180atttggaaaa aaatg 31952322DNAArtificial SequenceSynthetic 23tggaaataga ttcaggggtc at 222421DNAArtificial SequenceSynthetic 24cgggtgtacc tcactgactt c 212525DNAArtificial SequenceSynthetic 25tgtcttccga gagaaccagg ctccg 25
Patent applications by Herve E. Recipon, San Francisco, CA US
Patent applications by Jason Pluta, Mountain View, CA US
Patent applications by Roberto A. Macina, San Jose, CA US
Patent applications by Sei-Yu Chen, Foster City, CA US
Patent applications by Yongming Sun, San Jose, CA US
Patent applications in class Attached to antibody or antibody fragment or immunoglobulin; derivative
Patent applications in all subclasses Attached to antibody or antibody fragment or immunoglobulin; derivative